Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of chronic or non-chronic inflammatory eye diseases

ABSTRACT

The present invention refers to the use of protein kinase inhibitors and more specifically to the use of inhibitors of the protein kinase c-Jun amino terminal kinase, JNK inhibitor (poly-)peptides, chimeric peptides, or of nucleic acids encoding same as well as pharmaceutical compositions containing same, for the treatment of non-chronic or chronic inflammatory eye diseases, such as inflammatory diseases of the blephara, conjunctiva, cornea, sclera, the vitreous body, uvea, ciliary body, choroid, orbital bone, lacrimal gland, or iris, in particular wherein the inflammatory disease is selected from hordeolum, chalazion, conjunktivitis, keratitis, scieritis, episcleritis, endophthalmitis, panophtalmitis, irititis, uveitis, cyclitis, chorioiditis, orbital phlegmon, and myositis of the eye muscle etc.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

A computer readable text file, entitled“067802-5030-01_SequenceListing.txt,” created on or about 3 Sep. 2015,with a file size of about 62 kb contains the sequence listing for thisapplication and is hereby incorporated by reference in its entirety.

The present invention refers to the use of protein kinase inhibitors andmore specifically to the use of inhibitors of the protein kinase c-Junamino terminal kinase, JNK inhibitor (poly-)peptides, chimeric peptides,or of nucleic acids encoding same as well as pharmaceutical compositionscontaining same, for the treatment of non-chronic or chronicinflammatory eye diseases, such as hordeolum, chalazion, conjunktivitis,keratitis, scleritis, episcleritis, endophthalmitis, panophtalmitis,irititis, uveitis, cyclitis, chorioiditis, orbital phlegmon, and/ormyositis of the eye muscle, etc.

The number of ophthalmological (eye) diseases, particularly ofnon-chronic and chronic ophthalmological (eye) diseases represents aconsiderable challenge for the public health care systems.Ophthalmological diseases are diseases that pertain to the eye. Thepresent invention focuses on non-chronic or chronic inflammatory eyediseases. These include for example inflammatory diseases of theblephara, conjunctiva, cornea, sclera, the vitreous body, uvea, ciliarybody, choroid, orbital bone, lacrimal gland, iris, etc. Examples of suchinflammatory diseases are hordeolum, chalazion, conjunktivitis,keratitis, scleritis, episcleritis, endophthalmitis, panophtalmitis,irititis, uveitis, cyclitis, chorioiditis, orbital phlegmon, and/ormyositis of the eye muscle.

The c-Jun NH2-terminal kinases (JNKs) have been identified asstress-activated protein kinases that phosphorylate c-Jun on two sitesin its NH2-terminal activation domain. The JNK pathway is activated bycertain cytokines, mitogens, osmotic stress and irradiation. Thephosphorylation of the c-Jun component of the activator protein AP-1transcription factor results in pro-inflammatory cytokines production.During inflammation, leukocytes infiltration and rolling result from theearly activation of the vascular endothelium that releases importantchemotactic factors such as RANTES, IL-8, ICAM and VCAM. Infiltratingcells in turn release distinct sets of pro- or anti-inflammatoryproducts that contribute to tissue damages and inflammation. Many of thegene products involved in the inflammatory response are regulated by thetranscription factor activator protein-1 (AP-1), and the c-JunNH2-terminal kinase (JNK) pathway: COX-2, cyclooxygenase-2; IFN-g,interferon-gamma; iNOS, inducible nitric oxide synthase; TNF-a,tumor-necrosis factor-alpha; MCP-1, membrane cofactor protein-1; MIP-1,major intrinsic protein-1; IL-2, interleukin-2, . . . . Inlipopolysaccharide (LPS)-stimulated monocytes and tissue macrophages,TNF-a is produced through the JNK pathway activation and modulated byits inhibition.

JNK inhibitors have been therefore used in various models ofinflammation and shown to exert anti-inflammatory and beneficial effectsin inflammatory diseases such arthritis and asthma.

The object of the present invention is thus to provide alternative orimproved therapies, which allow new and preferably improved cure ofnon-chronic or chronic (inflammatory) eye diseases, such as hordeolum,chalazion, conjunktivitis, keratitis, scleritis, episcleritis,endophthalmitis, panophtalmitis, irititis, uveitis, cyclitis,chorioiditis, orbital phlegmon, myositis of the eye muscle, etc.

This object is solved by the use of a JNK inhibitor (poly-)peptidecomprising less than 150 amino acids in length for the preparation of apharmaceutical composition for treating non-chronic or chronicinflammatory eye diseases in a subject.

The term “non-chronic or chronic inflammatory eye disease” as usedherein typically denotes non-chronic or chronic inflammatory diseasesthat pertain to the eye. This includes diseases of the blephara,conjunctiva, cornea, sclera, the vitreous body, uvea, ciliary body,choroid, orbital bone, lacrimal gland, iris, etc. Preferably included inthis respect are hordeolum, chalazion, conjunktivitis, keratitis,scleritis, episcleritis, endophthalmitis, panophtalmitis, irititis,uveitis, cyclitis, chorioiditis, orbital phlegmon, myositis of the eyemuscle. Particularly preferred in the context of the present inventionis the treatment of uveitis, for example treatment of anterior uveitis,intermediate uveitis, posterior uveitis and panuveitis.

The present inventors surprisingly found, that JNK inhibitor(poly-)peptides s are particularly suitable for treating such chronic ornon-chronic inflammatory eye diseases in a subject. This was neitherobvious nor suggested by the prior art, even though JNK inhibitor(poly-)peptides in general have been known from the art.

In the context of the present invention, a JNK inhibitor (poly-)peptidemay be typically derived from a human or rat IB1 sequence, preferablyfrom an amino acid sequence as defined or encoded by any of sequencesaccording to SEQ ID NO: 102 (depicts the IB1 cDNA sequence from rat andits predicted amino acid sequence), SEQ ID NO: 103 (depicts the IB1protein sequence from rat encoded by the exon-intron boundary of therIB1 gene-splice donor), SEQ ID NO: 104 (depicts the IB1 proteinsequence from Homo sapiens), or SEQ ID NO: 105 (depicts the IB1 cDNAsequence from Homo sapiens), more preferably from an amino acid sequenceas defined or encoded by any of sequences according to SEQ ID NO: 104(depicts the IB1 protein sequence from Homo sapiens), or SEQ ID NO: 105(depicts the IB1 cDNA sequence from Homo sapiens), or from any fragmentsor variants thereof. In other words, the JNK inhibitor (poly-)peptidecomprises a fragment, variant, or variant of such fragment of a human orrat IB1 sequence. Human or rat IB sequences are defined or encoded,respectively, by the sequences according to SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO: 104 or SEQ ID NO: 105.

Preferably, such a JNK inhibitor (poly-)peptide as used herein comprisesa total length of less than 150 amino acid residues, preferably a rangeof 5 to 150 amino acid residues, more preferably 10 to 100 amino acidresidues, even more preferably 10 to 75 amino acid residues and mostpreferably a range of 10 to 50 amino acid residues, e.g. 10 to 30, 10 to20, or 10 to 15 amino acid residues.

More preferably, such a JNK inhibitor (poly-)peptide and the aboveranges may be selected from any of the above mentioned sequences, evenmore preferably from an amino acid sequence as defined according to SEQID NO: 104 or as encoded by SEQ ID NO: 105, even more preferably in theregion between nucleotides 420 and 980 of SEQ ID NO: 105 or amino acids105 and 291 of SEQ ID NO: 104, and most preferably in the region betweennucleotides 561 and 647 of SEQ ID NO: 105 or amino acids 152 and 180 ofSEQ ID NO: 104.

According to a particular embodiment, a JNK inhibitor (poly-)peptide asused herein typically binds JNK and/or inhibits the activation of atleast one JNK activated transcription factor, e.g. c-Jun or ATF2 (seee.g. SEQ ID NOs: 15 and 16, respectively) or Elk1.

Likewise, the JNK inhibitor (poly-)peptide as used herein preferablycomprises or consists of at least one amino acid sequence according toany one of SEQ ID NOs: 1 to 4, 13 to 20 and 33 to 100, or a fragment,derivative or variant thereof. More preferably, the JNK inhibitor(poly-) peptide as used herein may contain 1, 2, 3, 4 or even morecopies of an amino acid sequence according to SEQ ID NOs: 1 to 4, 13 to20 and 33 to 100, or a variant, fragment or derivative thereof. Ifpresent in more than one copy, these amino acid sequences according toSEQ ID NOs: 1 to 4, 13 to 20 and 33 to 100, or variants, fragments, orderivatives thereof as used herein may be directly linked with eachother without any linker sequence or via a linker sequence comprising 1to 10, preferably 1 to 5 amino acids. Amino acids forming the linkersequence are preferably selected from glycine or proline as amino acidresidues. More preferably, these amino acid sequences according to SEQID NOs: 1 to 4, 13 to 20 and 33 to 100, or fragments, variants orderivatives thereof, as used herein, may be separated by each other by ahinge of two, three or more proline residues.

The JNK inhibitor (poly-)peptides as used herein may be composed ofL-amino acids, D-amino acids, or a combination of both. Preferably, theJNK inhibitor (poly-)peptides as used herein comprise at least 1 or even2, preferably at least 3, 4 or 5, more preferably at least 6, 7, 8 or 9and even more preferably at least 10 or more D- and/or L-amino acids,wherein the D- and/or L-amino acids may be arranged in the JNK inhibitorsequences as used herein in a blockwise, a non-blockwise or in analternate manner.

According to one preferred embodiment the JNK inhibitor (poly-)peptidesas used herein may be exclusively composed of L-amino acids. The JNKinhibitor (poly-)peptides as used herein may then comprise or consist ofat least one native JNK inhibitor sequence” according to SEQ ID NO: 1 or3. In this context, the term “native” or “native JNK inhibitorsequence(s)” is referred to non-altered JNK inhibitor (poly-)peptidesequences according to any of SEQ ID NOs: 1 or 3, as used herein,entirely composed of L-amino acids.

Accordingly, the JNK inhibitor (poly-)peptide as used herein maycomprise or consist of at least one (native) amino acid sequenceNH₂—X_(n) ^(b)—X_(n) ^(a)-RPTTLXLXXXXXXXQD-X_(n) ^(b)—COOH (L-IB generic(s)) [SEQ ID NO: 3] and/or the JNK binding domain (JBDs) of IB1XRPTTLXLXXXXXXXQDS/TX (L-IB (generic)) [SEQ ID NO: 19]. In this context,each X typically represents an amino acid residue, preferably selectedfrom any (native) amino acid residue. X_(n) ^(a) typically representsone amino acid residue, preferably selected from any amino acid residueexcept serine or threonine, wherein n (the number of repetitions of X)is 0 or 1. Furthermore, each X_(n) ^(b) may be selected from any aminoacid residue, wherein n (the number of repetitions of X) is 0-5, 5-10,10-15, 15-20, 20-30 or more, provided that if n (the number ofrepetitions of X) is 0 for X_(n) ^(a), X_(n) ^(b) does preferably notcomprise a serine or threonine at its C-terminus, in order to avoid aserine or threonine at this position. Preferably, X_(n) ^(b) representsa contiguous stretch of peptide residues derived from SEQ ID NO: 1 or 3.X_(n) ^(a) and X_(n) ^(b) may represent either D or L amino acids.Additionally, the JNK inhibitor (poly-)peptide as used herein maycomprise or consist of at least one (native) amino acid sequenceselected from the group comprising the JNK binding domain of IB1DTYRPKRPTTLNLFPQVPRSQDT (L-IB1) [SEQ ID NO: 17]. More preferably, theJNK inhibitor (poly-)peptide as used herein further may comprise orconsist of at least one (native) amino acid sequenceNH₂—RPKRPTTLNLFPQVPRSQD-COOH (L-IB1(s)) [SEQ ID NO: 1]. Furthermore, theJNK inhibitor (poly-)peptide as used herein may comprise or consist ofat least one (native) amino acid sequence selected from the groupcomprising the JNK binding domain of IB1 L-IB1(s1)(NH₂-TLNLFPQVPRSQD-COOH, SEQ ID NO: 33); L-IB1(s2)(NH₂-TTLNLFPQVPRSQ-COOH, SEQ ID NO: 34); L-IB1(s3)(NH₂-PTTLNLFPQVPRS—COOH, SEQ ID NO: 35); L-IB1(s4)(NH₂—RPTTLNLFPQVPR—COOH, SEQ ID NO: 36); L-IB1(s5)(NH₂—KRPTTLNLFPQVP—COOH, SEQ ID NO: 37); L-IB1(s6)(NH₂—PKRPTTLNLFPQV—COOH, SEQ ID NO: 38); L-IB1(s7)(NH₂—RPKRPTTLNLFPQ-COOH, SEQ ID NO: 39); L-IB1(s8)(NH₂-LNLFPQVPRSQD-COOH, SEQ ID NO: 40); L-IB1(s9)(NH₂-TLNLFPQVPRSQ-COOH, SEQ ID NO: 41); L-IB1(s10)(NH₂-TTLNLFPQVPRS—COOH, SEQ ID NO: 42); L-IB1(s11)(NH₂-PTTLNLFPQVPR—COOH, SEQ ID NO: 43); L-IB1(s12)(NH₂—RPTTLNLFPQVP—COOH, SEQ ID NO: 44); L-IB1(s13)(NII₂-KRPTTLNLFPQV—COOH, SEQ ID NO: 45); L-IB1(s14)(NH₂-PKRPTTLNLFPQ-COOH, SEQ ID NO: 46); L-IB1(s15)(NH₂—RPKRPTTLNLFP—COOH, SEQ ID NO: 47); L-IB1(s16)(NH₂-NLFPQVPRSQD-COOH, SEQ ID NO: 48); L-IB1(s17) (NH₂-LNLFPQVPRSQ-COOH,SEQ ID NO: 49); L-IB1(s18) (NH₂-TLNLFPQVPRS—COOH, SEQ ID NO: 50);L-IB1(s19) (NH₂-TTLNLFPQVPR—COOH, SEQ ID NO: 51); L-IB1(s20)PTTLNLFPQVP—COOH, SEQ ID NO: 52); L-IB1(s21) (NH₂—RPTTLNLFPQV—COOH, SEQID NO: 53); L-IB1(s22) (NH₂—KRPTTLNLFPQ-COOH, SEQ ID NO: 54); L-IB1(s23)(NH₂-PKRPTTLNLFP—COOH, SEQ ID NO: 55); L-IB1(s24) (NH₂—RPKRPTTLNLF—COOH,SEQ ID NO: 56); L-IB1(s25) (NH₂-LFPQVPRSQD-COOH, SEQ ID NO: 57);L-IB1(s26) (NH₂-NLFPQVPRSQ-COOH, SEQ ID NO: 58); L-IB1(s27)(NH₂-LNLFPQVPRS—COOH, SEQ ID NO: 59); L-IB1(s28) (NH₂-TLNLFPQVPR—COOH,SEQ ID NO: 60); L-IB1(s29) (NH₂-TTLNLFPQVP—COOH, SEQ ID NO: 61);L-IB1(s30) (NH₂-PTTLNLFPQV—COOH, SEQ ID NO: 62); L-IB1(s31)(NH₂—RPTTLNLFPQ-COOH, SEQ ID NO: 63); L-IB1(s32) (NH₂—KRPTTLNLFP—COOH,SEQ ID NO: 64); L-IB1(s33) (NH₂-PKRPTTLNLF—COOH, SEQ ID NO: 65); andL-IB1(s34) (NH₂—RPKRPTTLNL-COOH, SEQ ID NO: 66).

Additionally, the JNK inhibitor (poly-)peptide as used herein maycomprise or consist of at least one (native) amino acid sequenceselected from the group comprising the (long) JNK binding domain (JBDs)of IB1 PGTGCGDTYRPKRPTTLNLFPQVPRSQDT (IB1-long) [SEQ ID NO: 13], the(long) JNK binding domain of IB2 IPSPSVEEPHKHRPTTLRLTTLGAQDS (IB2-long)[SEQ ID NO: 14], the JNK binding domain of c-JunGAYGYSNPKILKQSMTLNLADPVGNLKPH (c-Jun) [SEQ ID NO: 15], the JNK bindingdomain of ATF2 TNEDHLAVHKHKHEMTLKFGPARNDSVIV (ATF2) [SEQ ID NO: 16] (seee.g. FIGS. 1A-1C). In this context, an alignment revealed a partiallyconserved 8 amino acid sequence (see e.g. FIG. 1A) and a furthercomparison of the JBDs of IB1 and IB2 revealed two blocks of seven andthree amino acids that are highly conserved between the two sequences.

According to another preferred embodiment the JNK inhibitor(poly-)peptides as used herein may be composed in part or exclusively ofD-amino acids as defined above. More preferably, these JNK inhibitor(poly-)peptides composed of D-amino acids are non-native D retro-inversosequences of the above (native) JNK inhibitor sequences. The term“retro-inverso (poly-) peptides” refers to an isomer of a linear peptidesequence in which the direction of the sequence is reversed and thechirality of each amino acid residue is inverted (see e.g. Jameson etal., Nature, 368,744-746 (1994); Brady et al., Nature, 368,692-693(1994)). The advantage of combining D-enantiomers and reverse synthesisis that the positions of carbonyl and amino groups in each amide bondare exchanged, while the position of the side-chain groups at each alphacarbon is preserved. Unless specifically stated otherwise, it ispresumed that any given L-amino acid sequence or peptide as usedaccording to the present invention may be converted into an Dretro-inverso sequence or peptide by synthesizing a reverse of thesequence or peptide for the corresponding native L-amino acid sequenceor peptide.

The D retro-inverso (poly-)peptides as used herein and as defined abovehave a variety of useful properties. For example, D retro-inverso(poly-)peptides as used herein enter cells as efficiently as L-aminoacid sequences as used herein, whereas the D retro-inverso sequences asused herein are more stable than the corresponding L-amino acidsequences.

Accordingly, the JNK inhibitor (poly-)peptides as used herein maycomprise or consist of at least one D retro-inverso sequence accordingto the amino acid sequence NH₂—X_(n) ^(b)-DQXXXXXXXLXLTTPR—X_(n)^(a)—X_(n) ^(b)—COOH (D-IB1 generic (s)) [SEQ ID NO: 4] and/orXS/TDQXXXXXXXLXLTTPRX (D-IB (generic)) [SEQ ID NO: 20]. As used in thiscontext, X, X_(n) ^(a) and X_(n) ^(b) are as defined above (preferably,representing D amino acids), wherein X_(n) ^(b) preferably represents acontiguous stretch of residues derived from SEQ ID NO: 2 or 4.Additionally, the JNK inhibitor (poly-)peptides as used herein maycomprise or consist of at least one D retro-inverso sequence accordingto the amino acid sequence comprising the JNK binding domain (JBDs) ofIB1 TDQSRPVQPFLNLTTPRKPRYTD (D-IB1) [SEQ ID NO: 18]. More preferably,the JNK inhibitor (poly-)peptides as used herein may comprise or consistof at least one D retro-inverso sequence according to the amino acidsequence NH₂-DQSRPVQPFLNLTTPRKPR—COOH (D-IB1(s)) [SEQ ID NO: 2].Furthermore, the JNK inhibitor (poly-)peptides as used herein maycomprise or consist of at least one D retro-inverso sequence accordingto the amino acid sequence comprising the JNK binding domain (JBDs) ofIB1 D-IB1(s1) (NH₂-QPFLNLTTPRKPR—COOH, SEQ ID NO: 67); D-IB1(s2)(NH₂—VQPFLNLTTPRKP—COOH, SEQ ID NO: 68); D-IB1(s3)(NH₂—PVQPFLNLTTPRK—COOH, SEQ ID NO: 69); D-IB1(s4)(NH₂—RPVQPFLNLTTPR—COOH, SEQ ID NO: 70); D-M1(s5)(NH₂—SRPVQPFLNLTTP—COOH, SEQ ID NO: 71); D-IB1(s6)(NH₂-QSRPVQPFLNLTT-COOH, SEQ ID NO: 72); D-IB1(s7)(NH₂-DQSRPVQPFLNLT-COOH, SEQ ID NO: 73); D-IB1(s8)(NH₂—PFLNLTTPRKPR—COOH, SEQ ID NO: 74); D-IB1(s9)(NH₂-QPFLNLTTPRKP—COOH, SEQ ID NO: 75); D-IB1(s10)(NH₂—VQPFLNLTTPRK—COOH, SEQ ID NO: 76); D-IB1(s11)(NH₂—PVQPFLNLTTPR—COOH, SEQ ID NO: 77); D-IB1(s12)(NH₂—RPVQPFLNLTTP—COOH, SEQ ID NO: 78); D-IB1(s13)(NH₂—SRPVQPFLNLTT-COOH, SEQ ID NO: 79); D-IB1(s14)(NH₂-QSRPVQPFLNLT-COOH, SEQ ID NO: 80); D-IB1(s15)(NH₂-DQSRPVQPFLNL-COOH, SEQ ID NO: 81); D-IB1(s16)(NH₂—FLNLTTPRKPR—COOH, SEQ ID NO: 82); D-IB1(s17) (NH₂—PFLNLTTPRKP—COOH,SEQ ID NO: 83); D-IB1(s18) (NH₂-QPFLNLTTPRK—COOH, SEQ ID NO: 84);D-IB1(s19) (NH₂—VQPFLNLTTPR—COOH, SEQ ID NO: 85); D-IB1(s20)(NH₂—PVQPFLNLTTP—COOH, SEQ ID NO: 86); D-IB1(s21) (NH₂—RPVQPFLNLTT-COOH,SEQ ID NO: 87); D-IB1(s22) (NH₂—SRPVQPFLNLT-COOH, SEQ ID NO: 88);D-IB1(s23) (NH₂-QSRPVQPFLNL-COOH, SEQ ID NO: 89); D-IB1(s24)(NH₂-DQSRPVQPFLN—COOH, SEQ ID NO: 90); D-IB1(s25) (NH₂-DQSRPVQPFL-COOH,SEQ ID NO: 91); D-IB1(s26) (NH₂-QSRPVQPFLN-COOH, SEQ ID NO: 92);D-M1(s27) (NH₂—SRPVQPFLNL-COOH, SEQ ID NO: 93); D-IB1(s28)(NH₂—RPVQPFLNLT-COOH, SEQ ID NO: 94); D-IB1(s29) (NH₂—PVQPFLNLTT-COOH,SEQ ID NO: 95); D-IB1(s30) (NH₂—VQPFLNLTTP—COOH, SEQ ID NO: 96);D-IB1(s31) (NH₂-QPFLNLTTPR—COOH, SEQ ID NO: 97); D-IB1(s32)(NH₂—PFLNLTTPRK—COOH, SEQ ID NO: 98); D-IB1(s33) (NH₂—FLNLTTPRKP—COOH,SEQ ID NO: 99); and D-IB1(s34) (NH₂-LNLTTPRKPR—COOH, SEQ ID NO: 100).

The JNK inhibitor (poly-)peptides as used herein and as disclosed aboveare presented in Table 1 (SEQ ID NO:s 1-4, 13-20 and 33-100). The tablepresents the name of the JNK inhibitor (poly-) peptides/sequences asused herein, as well as their sequence identifier number, their length,and amino acid sequence. Furthermore, Table 1 shows sequences as well astheir generic formulas, e.g. for SEQ ID NO's: 1, 2, 5, 6, 9 and 11 andSEQ ID NO's: 3, 4, 7, 8, 10 and 12, respectively. Table 1 furthermorediscloses the chimeric sequences SEQ ID NOs: 9-12 and 23-32 (see below),L-IB1 sequences SEQ ID NOs: 33 to 66 and D-IB1 sequences SEQ ID NOs: 67to 100.

TABLE 1 SEQUENCE/PEPTIDE SEQ NAME ID NO AA SEQUENCE L-IB₁(s)   1 19RPKRPTTLNLFPQVPRSQD (NH₂-RPKRPTTLNLFPQVPRSQD-COOH) D-IB₁(s)   2 19DQSRPVQPFLNLTTPRKPR (NH₂-DQSRPVQPFLNLTTPRKPR-COOH) L-IB(generic)(s)   319 NH₂-X_(n) ^(b)-X_(n) ^(a)-RPTTLXLXXXXXXXQD-X_(n) ^(b)-COOHD-IB(generic)(s)   4 19 NH₂-X_(n) ^(b)-DQXXXXXXXLXLTTPR-X_(n) ^(a)-X_(n)^(b)-COOH L-TAT   5 10 GRKKRRQRRR (NH₂-GRKKRRQRRR-COOH) D-TAT   6 10RRRQRRKKRG (NH₂-RRRQRRKKRG-COOH) L-generic-TAT(s)   7 11 NH₂-X_(n)^(b)-RKKRRQRRR-X_(n) ^(b)-COOH D-generic-TAT(s)   8 11 NH₂-X_(n)^(b)-RRRQRRKKR-X_(n) ^(b)-COOH L-TAT-IB₁(s)   9 31GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQD(NH₂-GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQD-COOH) L-TAT-IB(generic)(s)  10 29NH₂-X_(n) ^(b)-RKKRRQRRR-X_(n) ^(b)-X_(n) ^(a)-RPTTLXLXXXXXXXQD-X_(n)^(b)-COOH D-TAT-IB₁(s)  11 31 DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG(NH₂-DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG-COOH) D-TAT-IB(generic)(s)  12 29(NH₂-X_(n) ^(b)-DQXXXXXXXLXLTTPR-X_(n) ^(a)-X_(n) ^(b)-RRRQRRKKR-X_(n)^(b)-COOH IB1-long  13 29 PGTGCGDTYRPKRPTTLNLFPQVPRSQDT(NH₂-PGTGCGDTYRPKRPTTLNLFPQVPRSQDT-COOH) IB2-long  14 27IPSPSVEEPHKHRPTTLRLTTLGAQDS (NH₂-IPSPSVEEPHKHRPTTLRLTTLGAQDS-COOH) c-Jun 15 29 GAYGYSNPKILKQSMTLNLADPVGNLKPH(NH₂-GAYGYSNPKILKQSMTLNLADPVGNLKPH-COOH) ATF₂  16 29TNEDHLAVHKHKHEMTLKFGPARNDSVIV (NH₂-TNEDHLAVHKHKHEMTLKFGPARNDSVIV-COOH)L-IB₁  17 23 DTYRPKRPTTLNLFPQVPRSQDT (NH₂-DTYRPKRPTTLNLFPQVPRSQDT-COOH)D-IB₁  18 23 TDQSRPVQPFLNLTTPRKPRYTD (NH₂-TDQSRPVQPFLNLTTPRKPRYTD-COOH)L-IB(generic)  19 19 XRPTTLXLXXXXXXXQDS/TX(NH₂-XRPTTLXLXXXXXXXQDS/TX-COOH) D-IB(generic)  20 19XS/TDQXXXXXXXLXLTTPRX (NH₂-XS/TDQXXXXXXXLXLTTPRX-COOH) L-generic-TAT  2117 XXXXRKKRRQRRRXXXX (NH₂-XXXXRKKRRQRRRXXXX-COOH) D-generic-TAT  22 17XXXXRRRQRRKKRXXXX (NH₂-XXXXRRRQRRKKRXXXX-COOH) L-TAT-IB₁  23 35GRKKRRQRRRPPDTYRPKRPTTLNLFPQVPRSQDT(NH₂-GRKKRRQRRRPPDTYRPKRPTTLNLFPQVPRSQDT-COOH) L-TAT-IB(generic)  24 42XXXXXXXRKKRRQRRRXXXXXXXXRPTTLXLXXXXXXXQDS/TX (NH₂-XXXXXXXRKKRRQRRRXXXXXXXXRPTTLXLXXXXXXXQDS/TX-COOH) D-TAT-IB₁  25 35TDQSRPVQPFLNLTTPRKPRYTDPPRRRQRRKKRG(NH₂-TDQSRPVQPFLNLTTPRKPRYTDPPRRRQRRKKRG-COOH) D-TAT-IB(generic)  26 42XT/SDQXXXXXXXLXLTTPRXXXXXXXXRRRQRRKKRXXXXXXX (NH₂-XT/SDQXXXXXXXLXLTTPRXXXXXXXXRRRQRRKKRXXXXXXX- COOH) L-TAT-IB₁(s1)  27 30RKKRRQRRRPPRPKRPTTLNLFPQVPRSQD (NH₂-RKKRRQRRRPPRPKRPTTLNLFPQVPRSQD-COOH)L-TAT-IB₁(s2)  28 30 GRKKRRQRRRX_(n) ^(c)RPKRPTTLNLFPQVPRSQD(NH₂-GRKKRRQRRRX_(n) ^(c)RPKRPTTLNLFPQVPRSQD-COOH) L-TAT-IB₁(s3)  29 29RKKRRQRRRX_(n) ^(c)RPKRPTTLNLFPQVPRSQD (NH₂-RKKRRQRRRX_(n)^(c)RPKRPTTLNLFPQVPRSQD-COOH) D-TAT-IB₁(s1)  30 30DQSRPVQPFLNLTTPRKPRPPRRRQRRKKR (NH₂-DQSRPVQPFLNLTTPRKPRPPRRRQRRKKR-COOH)D-TAT-IB₁(s2)  31 30 DQSRPVQPFLNLTTPRKPRX_(n) ^(c)RRRQRRKKRG(NH₂-DQSRPVQPFLNLTTPRKPRX_(n) ^(c)RRRQRRKKRG-COOH) D-TAT-IB₁(s3)  32 29DQSRPVQPFLNLTTPRKPRX_(n) ^(c)RRRQRRKKR (NH₂-DQSRPVQPFLNLTTPRKPRX_(n)^(c)RRRQRRKKR-COOH) L-IB₁(s1)  33 13 TLNLFPQVPRSQD(NH₂-TLNLFPQVPRSQD-COOH) L-IB₁(s2)  34 13 TTLNLFPQVPRSQ(NH₂-TTLNLFPQVPRSQ-COOH) L-IB₁(s3)  35 13 PTTLNLFPQVPRS(NH₂-PTTLNLFPQVPRS-COOH) L-IB₁(s4)  36 13 RPTTLNLFPQVPR(NH₂-RPTTLNLFPQVPR-COOH) L-IB₁(s5)  37 13 KRPTTLNLFPQVP(NH₂-KRPTTLNLFPQVP-COOH) L-IB₁(s6)  38 13 PKRPTTLNLFPQV(NH₂-PKRPTTLNLFPQV-COOH) L-IB₁(s7)  39 13 RPKRPTTLNLFPQ(NH₂-RPKRPTTLNLFPQ-COOH) L-IB₁(s8)  40 12 LNLFPQVPRSQD(NH₂-LNLFPQVPRSQD-COOH) L-IB₁(s9)  41 12 TLNLFPQVPRSQ(NH₂-TLNLFPQVPRSQ-COOH) L-IB₁(s10)  42 12 TTLNLFPQVPRS(NH₂-TTLNLFPQVPRS-COOH) L-IB₁(s11)  43 12 PTTLNLFPQVPR(NH₂-PTTLNLFPQVPR-COOH) L-IB₁(s12)  44 12 RPTTLNLFPQVP(NH₂-RPTTLNLFPQVP-COOH) L-IB₁(s13)  45 12 KRPTTLNLFPQV(NH₂-KRPTTLNLFPQV-COOH) L-IB₁(s14)  46 12 PKRPTTLNLFPQ(NH₂-PKRPTTLNLFPQ-COOH) L-IB₁(s15)  47 12 RPKRPTTLNLFP(NH₂-RPKRPTTLNLFP-COOH) L-IB₁(s16)  48 11 NLFPQVPRSQD(NH₂-NLFPQVPRSQD-COOH) L-IB₁(s17)  49 11 LNLFPQVPRSQ(NH₂-LNLFPQVPRSQ-COOH) L-IB₁(s18)  50 11 TLNLFPQVPRS(NH₂-TLNLFPQVPRS-COOH) L-IB₁(s19)  51 11 TTLNLFPQVPR(NH₂-TTLNLFPQVPR-COOH) L-IB₁(s20)  52 11 PTTLNLFPQVP(NH₂-PTTLNLFPQVP-COOH) L-IB₁(s21)  53 11 RPTTLNLFPQV(NH₂-RPTTLNLFPQV-COOH) L-IB₁(s22)  54 11 KRPTTLNLFPQ(NH₂-KRPTTLNLFPQ-COOH) L-IB₁(s23)  55 11 PKRPTTLNLFP(NH₂-PKRPTTLNLFP-COOH) L-IB₁(s24)  56 11 RPKRPTTLNLF(NH₂-RPKRPTTLNLF-COOH) L-IB₁(s25)  57 10 LFPQVPRSQD(NH₂-LFPQVPRSQD-COOH) L-IB₁(s26)  58 10 NLFPQVPRSQ (NH₂-NLFPQVPRSQ-COOH)L-IB₁(s27)  59 10 LNLFPQVPRS (NH₂-LNLFPQVPRS-COOH) L-IB₁(s28)  60 10TLNLFPQVPR (NH₂-TLNLFPQVPR-COOH) L-IB₁(s29)  61 10 TTLNLFPQVP(NH₂-TTLNLFPQVP-COOH) L-IB₁(s30)  62 10 PTTLNLFPQV (NH₂-PTTLNLFPQV-COOH)L-IB₁(s31)  63 10 RPTTLNLFPQ (NH₂-RPTTLNLFPQ-COOH) L-IB₁(s32)  64 10KRPTTLNLFP (NH₂-KRPTTLNLFP-COOH) L-IB₁(s33)  65 10 PKRPTTLNLF(NH₂-PKRPTTLNLF-COOH) L-IB₁(s34)  66 10 RPKRPTTLNL (NH₂-RPKRPTTLNL-COOH)D-IB₁(s1)  67 13 QPFLNLTTPRKPR (NH₂-QPFLNLTTPRKPR-COOH) D-IB₁(s2)  68 13VQPFLNLTTPRKP (NH₂-VQPFLNLTTPRKP-COOH) D-IB₁(s3)  69 13 PVQPFLNLTTPRK(NH₂-PVQPFLNLTTPRK-COOH) D-IB₁(s4)  70 13 RPVQPFLNLTTPR(NH₂-RPVQPFLNLTTPR-COOH) D-IB₁(s5)  71 13 SRPVQPFLNLTTP(NH₂-SRPVQPFLNLTTP-COOH) D-IB₁(s6)  72 13 QSRPVQPFLNLTT(NH₂-QSRPVQPFLNLTT-COOH) D-IB₁(s7)  73 13 DQSRPVQPFLNLT(NH₂-DQSRPVQPFLNLT-COOH) D-IB₁(s8)  74 12 PFLNLTTPRKPR(NH₂-PFLNLTTPRKPR-COOH) D-IB₁(s9)  75 12 QPFLNLTTPRKP(NH₂-QPFLNLTTPRKP-COOH) D-IB₁(s10)  76 12 VQPFLNLTTPRK(NH₂-VQPFLNLTTPRK-COOH) D-IB₁(s11)  77 12 PVQPFLNLTTPR(NH₂-PVQPFLNLTTPR-COOH) D-IB₁(s12)  78 12 RPVQPFLNLTTP(NH₂-RPVQPFLNLTTP-COOH) D-IB₁(s13)  79 12 SRPVQPFLNLTT(NH₂-SRPVQPFLNLTT-COOH) D-IB₁(s14)  80 12 QSRPVQPFLNLT(NH₂-QSRPVQPFLNLT-COOH) D-IB₁(s15)  81 12 DQSRPVQPFLNL(NH₂-DQSRPVQPFLNL-COOH) D-IB₁(s16)  82 11 FLNLTTPRKPR(NH₂-FLNLTTPRKPR-COOH) D-IB₁(s17)  83 11 PFLNLTTPRKP(NH₂-PFLNLTTPRKP-COOH) D-IB₁(s18)  84 11 QPFLNLTTPRK(NH₂-QPFLNLTTPRK-COOH) D-IB₁(s19)  85 11 VQPFLNLTTPR(NH₂-VQPFLNLTTPR-COOH) D-IB₁(s20)  86 11 PVQPFLNLTTP(NH₂-PVQPFLNLTTP-COOH) D-IB₁(s21)  87 11 RPVQPFLNLTT(NH₂-RPVQPFLNLTT-COOH) D-IB₁(s22)  88 11 SRPVQPFLNLT(NH₂-SRPVQPFLNLT-COOH) D-IB₁(s23)  89 11 QSRPVQPFLNL(NH₂-QSRPVQPFLNL-COOH) D-IB₁(s24)  90 11 DQSRPVQPFLN(NH₂-DQSRPVQPFLN-COOH) D-IB₁(s25)  91 10 DQSRPVQPFL(NH₂-DQSRPVQPFL-COOH) D-IB₁(s26)  92 10 QSRPVQPFLN (NH₂-QSRPVQPFLN-COOH)D-IB₁(s27)  93 10 SRPVQPFLNL (NH₂-SRPVQPFLNL-COOH) D-IB₁(s28)  94 10RPVQPFLNLT (NH₂-RPVQPFLNLT-COOH) D-IB₁(s29)  95 10 PVQPFLNLTT(NH₂-PVQPFLNLTT-COOH) D-IB₁(s30)  96 10 VQPFLNLTTP (NH₂-VQPFLNLTTP-COOH)D-IB₁(s31)  97 10 QPFLNLTTPR (NH₂-QPFLNLTTPR-COOH) D-IB₁(s32)  98 10PFLNLTTPRK (NH₂-PFLNLTTPRK-COOH) D-IB₁(s33)  99 10 FLNLTTPRKP(NH₂-FLNLTTPRKP-COOH) D-IB₁(s34) 100 10 LNLTTPRKPR (NH₂-LNLTTPRKPR-COOH)

It will be understood by a person skilled in the art that a givensequence herein which is composed exclusively of D-amino acids isidentified by “D-name”. For example, SEQ ID NO:100 has thesequence/peptide name “D-IB1 (s34)”. The given amino acid sequence isLNLTTPRKPR (SEQ ID NO:100). However, all amino acids are here D-aminoacids.

It will be also understood by a person skilled in the art that the terms“entirely composed of L-amino acids”; “exclusively composed of D-aminoacids” “entirely composed of D-amino acids” and/or “exclusively composedof D-amino acids” and the like refer to sequences which need not (butmay) exclude the presence of glycine residues. Glycine is the only aminoacid which is non-chiral. Therefore, the terms “entirely composed ofL-amino acids”; “exclusively composed of D-amino acids” “entirelycomposed of D-amino acids” and/or “exclusively composed of D-aminoacids” are intended to make clear that L-amino acids or D-amino acids,respectively, are used where possible. Nevertheless, if presence of aglycine is necessary or favored at a given position in the amino acidsequence, then it may remain there. A good example is L-TAT (SEQ IDNO:5). As used herein said sequence is considered to be exclusivelycomposed of L-amino acids “although” said sequence comprises a nonchiral glycine residue. Likewise, D-TAT (SEQ ID NO:6), as used herein,may be considered to be exclusively composed of D-amino acids “although”said sequence comprises a non chiral glycine residue.

According to another preferred embodiment, the JNK inhibitor(poly-)peptide as used herein comprises or consists of at least onevariant, fragment and/or derivative of the above defined native ornon-native amino acid sequences according to SEQ ID NOs: 1-4, 13-20 and33-100.

Preferably, these variants, fragments and/or derivatives retainbiological activity of the above disclosed native or non-native JNKinhibitor (poly-)peptides as used herein, particularly of native ornon-native amino acid sequences according to SEQ ID NOs: 1-4, 13-20 and33-100, i.e. binding JNK and/or inhibiting the activation of at leastone JNK activated transcription factor, e.g. c-Jun, ATF2 or Elk1.Functionality may be tested by various tests, e.g. binding tests of thepeptide to its target molecule or by biophysical methods, e.g.spectroscopy, computer modeling, structural analysis, etc. Particularly,an JNK inhibitor (poly-)peptide or variants, fragments and/orderivatives thereof as defined above may be analyzed by hydrophilicityanalysis (see e.g. Hopp and Woods, 1981. Proc Natl Acad Sci USA 78:3824-3828) that can be utilized to identify the hydrophobic andhydrophilic regions of the peptides, thus aiding in the design ofsubstrates for experimental manipulation, such as in bindingexperiments, or for antibody synthesis. Secondary structural analysismay also be performed to identify regions of an JNK inhibitor(poly-)peptide or of variants, fragments and/or derivatives thereof asused herein that assume specific structural motifs (see e.g. Chou andFasman, 1974, Biochem 13: 222-223). Manipulation, translation, secondarystructure prediction, hydrophilicity and hydrophobicity profiles, openreading frame prediction and plotting, and determination of sequencehomologies can be accomplished using computer software programsavailable in the art. Other methods of structural analysis include, e.g.X-ray crystallography (see e.g. Engstrom, 1974. Biochem Exp Biol 11:7-13), mass spectroscopy and gas chromatography (see e.g. METHODS INPROTEIN SCIENCE, 1997, J. Wiley and Sons, New York, N.Y.) and computermodeling (see e.g. Fletterick and Zoller, eds., 1986. Computer Graphicsand Molecular Modeling, In: CURRENT COMMUNICATIONS IN MOLECULAR BIOLOGY,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) may alsobe employed.

Accordingly, the JNK inhibitor (poly-)peptide as used herein maycomprise or consist of at least one variant of (native or non-native)amino acid sequences according to SEQ ID NOs: 1-4, 13-20 and 33-100. Inthe context of the present invention, a “variant of a (native ornon-native) amino acid sequence according to SEQ ID NOs: 1-4, 13-20 and33-100” is preferably a sequence derived from any of the sequencesaccording to SEQ ID NOs: 1-4, 13-20 and 33-100, wherein the variantcomprises amino acid alterations of the amino acid sequences accordingto SEQ ID NOs: 1-4, 13-20 and 33-100. Such alterations typicallycomprise 1 to 20, preferably 1 to 10 and more preferably 1 to 5substitutions, additions and/or deletions of amino acids according toSEQ ID NOs: 1-4, 13-20 and 33-100, wherein the variant exhibits asequence identity with any of the sequences according to SEQ ID NOs:1-4, 13-20 and 33-100 of at least about 30%, 50%, 70%, 80%, 90%, 95%,98% or even at least about 99%.

If variants of (native or non-native) amino acid sequences according toSEQ ID NOs: 1-4, 13-20 and 33-100 as defined above and used herein areobtained by substitution of specific amino acids, such substitutionspreferably comprise conservative amino acid substitutions. Conservativeamino acid substitutions may include synonymous amino acid residueswithin a group which have sufficiently similar physicochemicalproperties, so that a substitution between members of the group willpreserve the biological activity of the molecule (see e.g. Grantham, R.(1974), Science 185, 862-864). It is evident to the skilled person thatamino acids may also be inserted and/or deleted in the above-definedsequences without altering their function, particularly if theinsertions and/or deletions only involve a few amino acids, e.g. lessthan twenty, and preferably less than ten, and do not remove or displaceamino acids which are critical to functional activity. Moreover,substitutions shall be avoided in variants as used herein, which lead toadditional threonines at amino acid positions which are accessible for aphosphorylase, preferably a kinase, in order to avoid inactivation ofthe JNK-inhibitor (poly-)peptide as used herein or of the chimericpeptide as used herein in vivo or in vitro.

Preferably, synonymous amino acid residues, which are classified intothe same groups and are typically exchangeable by conservative aminoacid substitutions, are defined in Table 2.

TABLE 2 Preferred Groups of Synonymous Amino Acid Residues Amino AcidSynonymous Residue Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, HisLeu Ile, Phe, Tyr, Met, Val, Leu Pro Gly, Ala, (Thr), Pro Thr Pro, Ser,Ala, Gly, His, Gln, Thr Ala Gly, Thr, Pro, Ala Val Met, Tyr, Phe, Ile,Leu, Val Gly Ala, (Thr), Pro, Ser, Gly Ile Met, Tyr, Phe, Val, Leu, IlePhe Trp, Met, Tyr, Ile, Val, Leu, Phe Tyr Trp, Met, Phe, Ile, Val, Leu,Tyr Cys Ser, Thr, Cys His Glu, Lys, Gln, Thr, Arg, His Gln Glu, Lys,Asn, His, (Thr), Arg, Gln Asn Gln, Asp, Ser, Asn Lys Glu, Gln, His, Arg,Lys Asp Glu, Asn, Asp Glu Asp, Lys, Asn, Gln, His, Arg, Glu Met Phe,Ile, Val, Leu, Met Trp Trp

A specific form of a variant of SEQ ID NOs: 1-4, 13-20 and 33-100 asused herein is a fragment of the (native or non-native) amino acidsequences according to SEQ ID NOs: 1, 1-4, 13-20 and 33-100″ as usedherein, which is typically altered by at least one deletion as comparedto SEQ ID NOs 1-4, 13-20 and 33-100. Preferably, a fragment comprises atleast 4 contiguous amino acids of any of SEQ ID NOs: 1-4, 13-20 and33-100, a length typically sufficient to allow for specific recognitionof an epitope from any of these sequences. Even more preferably, thefragment comprises 4 to 18, 4 to 15, or most preferably 4 to 10contiguous amino acids of any of SEQ ID NOs: 1-4, 13-20 and 33-100,wherein the lower limit of the range may be 4, or 5, 6, 7, 8, 9, or 10.Deleted amino acids may occur at any position of SEQ ID NOs: 1-4, 13-20and 33-100, preferably N- or C-terminally.

Furthermore, a fragment of the (native or non-native) amino acidsequences according to SEQ ID NOs: 1-4, 13-20 and 33-100, as describedabove, may be defined as a sequence sharing a sequence identity with anyof the sequences according to SEQ ID NOs: 1-4, 13-20 and 33-100 as usedherein of at least about 30%, 50%, 70%, 80%, 90%, 95%, 98%, or even 99%.

The JNK inhibitor (poly-)peptides/sequences as used herein may furthercomprise or consist of at least one derivative of (native or non-native)amino acid sequences according to SEQ ID NOs: 1-4, 13-20 and 33-100 asdefined above. In this context, a “derivative of an (native ornon-native) amino acid sequence according to SEQ ID NOs: 1-4, 13-20 and33-100” is preferably an amino acid sequence derived from any of thesequences according to SEQ ID NOs: 1-4, 13-20 and 33-100, wherein thederivative comprises at least one modified L- or D-amino acid (formingnon-natural amino acid(s)), preferably 1 to 20, more preferably 1 to 10,and even more preferably 1 to 5 modified L- or D-amino acids.Derivatives of variants or fragments also fall under the scope of thepresent invention.

“A modified amino acid” in this respect may be any amino acid which isaltered e.g. by different glycosylation in various organisms, byphosphorylation or by labeling specific amino acids. Such a label isthen typically selected from the group of labels comprising:

-   -   (i) radioactive labels, i.e. radioactive phosphorylation or a        radioactive label with sulphur, hydrogen, carbon, nitrogen,        etc.;    -   (ii) colored dyes (e.g. digoxygenin, etc.);    -   (iii) fluorescent groups (e.g. fluorescein, etc.);    -   (iv) chemolumminescent groups;    -   (v) groups for immobilization on a solid phase (e.g. IIis-tag,        biotin, strep-tag, flag-tag, antibodies, antigen, etc.); and    -   (vi) a combination of labels of two or more of the labels        mentioned under (i) to (v).

In the above context, an amino acid sequence having a sequence “sharinga sequence identity” of at least, for example, 95% to a query amino acidsequence of the present invention, is intended to mean that the sequenceof the subject amino acid sequence is identical to the query sequenceexcept that the subject amino acid sequence may include up to five aminoacid alterations per each 100 amino acids of the query amino acidsequence. In other words, to obtain an amino acid sequence having asequence of at least 95% identity to a query amino acid sequence, up to5% (5 of 100) of the amino acid residues in the subject sequence may beinserted or substituted with another amino acid or deleted.

For sequences without exact correspondence, a “% identity” of a firstsequence may be determined with respect to a second sequence. Ingeneral, these two sequences to be compared are aligned to give amaximum correlation between the sequences. This may include inserting“gaps” in either one or both sequences, to enhance the degree ofalignment. A % identity may then be determined over the whole length ofeach of the sequences being compared (so-called global alignment), thatis particularly suitable for sequences of the same or similar length, orover shorter, defined lengths (so-called local alignment), that is moresuitable for sequences of unequal length.

Methods for comparing the identity and homology of two or moresequences, particularly as used herein, are well known in the art. Thusfor instance, programs available in the Wisconsin Sequence AnalysisPackage, version 9.1 (Devereux et al., 1984, Nucleic Acids Res. 12,387-395.), for example the programs BESTFIT and GAP, may be used todetermine the % identity between two polynucleotides and the % identityand the % homology between two polypeptide sequences. BESTFIT uses the“local homology” algorithm of (Smith and Waterman (1981), J. Mol. Biol.147, 195-197.) and finds the best single region of similarity betweentwo sequences. Other programs for determining identity and/or similaritybetween sequences are also known in the art, for instance the BLASTfamily of programs (Altschul et al., 1990, J. Mol. Biol. 215, 403-410),accessible through the home page of the NCBI at world wide web sitencbi.nlm.nih.gov) and FASTA (Pearson (1990), Methods Enzymol. 183,63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. U. S. A 85,2444-2448.).

JNK-inhibitor (poly-)peptides/sequences as used according to the presentinvention and as defined above may be obtained or produced by methodswell-known in the art, e.g. by chemical synthesis or by geneticengineering methods as discussed below. For example, a peptidecorresponding to a portion of an JNK inhibitor sequence as used hereinincluding a desired region of said JNK inhibitor sequence, or thatmediates the desired activity in vitro or in vivo, may be synthesized byuse of a peptide synthesizer.

JNK inhibitor (poly-)peptide as used herein and as defined above, may befurthermore be modified by a trafficking (poly-)peptide, allowing theJNK inhibitor (poly-)peptide as used herein and as defined above to betransported effectively into the cells. Such modified JNK inhibitor(poly-)peptides are preferably provided and used as chimeric(poly-)peptides.

According to a second aspect the present invention therefore providesthe use of a chimeric (poly-) peptide including at least one firstdomain and at least one second domain, for the preparation of apharmaceutical composition for treating non-chronic or chronicinflammatory eye diseases in a subject, wherein the first domain of thechimeric peptide comprises a trafficking sequence, while the seconddomain of the chimeric (poly-)peptide comprises an JNK inhibitorsequence as defined above, preferably of any of sequences according toSEQ ID NO: 1-4, 13-20 and 33-100 or a derivative or a fragment thereof.

Typically, chimeric (poly-)peptides as used according to the presentinvention have a length of at least 25 amino acid residues, e.g. 25 to250 amino acid residues, more preferably 25 to 200 amino acid residues,even more preferably 25 to 150 amino acid residues, 25 to 100 and mostpreferably amino acid 25 to 50 amino acid residues.

As a first domain the chimeric (poly-)peptide as used herein preferablycomprises a trafficking sequence, which is typically selected from anysequence of amino acids that directs a peptide (in which it is present)to a desired cellular destination. Thus, the trafficking sequence, asused herein, typically directs the peptide across the plasma membrane,e.g. from outside the cell, through the plasma membrane, and into thecytoplasm. Alternatively, or in addition, the trafficking sequence maydirect the peptide to a desired location within the cell, e.g. thenucleus, the ribosome, the endoplasmic reticulum (ER), a lysosome, orperoxisome, by e.g. combining two components (e.g. a component for cellpermeability and a component for nuclear location) or by one singlecomponent having e.g. properties of cell membrane transport and targetede.g. intranuclear transport. The trafficking sequence may additionallycomprise another component, which is capable of binding a cytoplasmiccomponent or any other component or compartment of the cell (e.g.endoplasmic reticulum, mitochondria, gloom apparatus, lysosomalvesicles). Accordingly, e.g. the trafficking sequence of the firstdomain and the JNK inhibitor sequence of the second domain may belocalized in the cytoplasm or any other compartment of the cell. Thisallows to determine localization of the chimeric peptide in the cellupon uptake.

Preferably, the trafficking sequence (being included in the first domainof the chimeric peptide as used herein) has a length of 5 to 150 aminoacid sequences, more preferably a length of 5 to 100 and most preferablya length of from 5 to 50, 5 to 30 or even 5 to 15 amino acids.

More preferably, the trafficking sequence (contained in the first domainof the chimeric peptide as used herein) may occur as a continuous aminoacid sequence stretch in the first domain. Alternatively, thetrafficking sequence in the first domain may be splitted into two ormore fragments, wherein all of these fragments resemble the entiretrafficking sequence and may be separated from each other by 1 to 10,preferably 1 to 5 amino acids, provided that the trafficking sequence assuch retains its carrier properties as disclosed above. These aminoacids separating the fragments of the trafficking sequence may e.g. beselected from amino acid sequences differing from the traffickingsequence. Alternatively, the first domain may contain a traffickingsequence composed of more than one component, each component with itsown function for the transport of the cargo JNK inhibitor sequence ofthe second domain to e.g. a specific cell compartment.

The trafficking sequence as defined above may be composed of L-aminoacids, D-amino acids, or a combination of both. Preferably, thetrafficking sequences (being included in the first domain of thechimeric peptide as used herein) may comprise at least 1 or even 2,preferably at least 3, 4 or 5, more preferably at least 6, 7, 8 or 9 andeven more preferably at least 10 or more D- and/or L-amino acids,wherein the D- and/or L-amino acids may be arranged in the JNKtrafficking sequences in a blockwise, a non-blockwise or in an alternatemanner.

According to one alternative embodiment, the trafficking sequence of thechimeric (poly-)peptide as used herein may be exclusively composed ofL-amino acids. More preferably, the trafficking sequence of the chimericpeptide as used herein comprises or consists of at least one “native”trafficking sequence as defined above. In this context, the term“native” is referred to non-altered trafficking sequences, entirelycomposed of L-amino acids.

According to another alternative embodiment the trafficking sequence ofthe chimeric (poly-) peptide as used herein may be exclusively composedof D-amino acids. More preferably, the trafficking sequence of thechimeric peptide as used herein may comprise a D retro-inverso peptideof the sequences as presented above.

The trafficking sequence of the first domain of the chimeric(poly-)peptide as used herein may be obtained from naturally occurringsources or can be produced by using genetic engineering techniques orchemical synthesis (see e.g. Sambrook, J., Fritsch, E. F., Maniatis, T.(1989) Molecular cloning: A laboratory manual. 2nd edition. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

Sources for the trafficking sequence of the first domain may be employedincluding, e.g. native proteins such as e.g. the TAT protein (e.g. asdescribed in U.S. Pat. Nos. 5,804,604 and 5,674,980, each of thesereferences being incorporated herein by reference), VP22 (described ine.g. WO 97/05265; Elliott and O'Hare, Cell 88: 223-233 (1997)),non-viral proteins (Jackson et al, Proc. Natl. Acad. Sci. USA 89:10691-10695 (1992)), trafficking sequences derived from Antennapedia(e.g. the antennapedia carrier sequence) or from basic peptides, e.g.peptides having a length of 5 to 15 amino acids, preferably 10 to 12amino acids and comprising at least 80%, more preferably 85% or even 90%basic amino acids, such as e.g. arginine, lysine and/or histidine.Furthermore, variants, fragments and derivatives of one of the nativeproteins used as trafficking sequences are disclosed herewith. Withregard to variants, fragments and derivatives it is referred to thedefinition given above for JNK inhibitor sequences as used herein.Variants, fragments as well as derivatives are correspondingly definedas set forth above for JNK inhibitor sequences as used herein.Particularly, in the context of the trafficking sequence, a variant orfragment or derivative may be defined as a sequence sharing a sequenceidentity with one of the native proteins used as trafficking sequencesas defined above of at least about 30%, 50%, 70%, 80%, 90%, 95%, 98%, oreven 99%.

In a preferred embodiment of the chimeric (poly-)peptide as used herein,the trafficking sequence of the first domain comprises or consists of asequence derived from the human immunodeficiency virus (HIV) 1 TATprotein, particularly some or all of the 86 amino acids that make up theTAT protein.

For a trafficking sequence (being included in the first domain of thechimeric peptide as used herein), partial sequences of the full-lengthTAT protein may be used forming a functionally effective fragment of aTAT protein, i.e. a TAT peptide that includes the region that mediatesentry and uptake into cells. As to whether such a sequence is afunctionally effective fragment of the TAT protein can be determinedusing known techniques (see e.g. Franked et al., Proc. Natl. Acad. Sci,USA 86: 7397-7401 (1989)). Thus, the trafficking sequence in the firstdomain of the chimeric peptide as used herein may be derived from afunctionally effective fragment or portion of a TAT protein sequencethat comprises less than 86 amino acids, and which exhibits uptake intocells, and optionally the uptake into the cell nucleus. More preferably,partial sequences (fragments) of TAT to be used as carrier to mediatepermeation of the chimeric peptide across the cell membrane, areintended to comprise the basic region (amino acids 48 to 57 or 49 to 57)of full-length TAT.

According to a more preferred embodiment, the trafficking sequence(being included in the first domain of the chimeric peptide as usedherein) may comprise or consist of an amino acid sequence containing TATresidues 48-57 or 49 to 57, and most preferably a generic TAT sequenceNH₂—X_(n) ^(b)—RKKRRQRRR-X_(n) ^(b)—COOH (L-generic-TAT (s)) [SEQ ID NO:7] and/or XXXXRKKRRQ RRRXXXX (L-generic-TAT) [SEQ ID NO: 21], wherein Xor X_(n) ^(b) is as defined above. Furthermore, the number of “X_(n)^(b)” residues in SEQ ID NOs:8 is not limited to the one depicted, andmay vary as described above. Alternatively, the trafficking sequencebeing included in the first domain of the chimeric peptide as usedherein may comprise or consist of a peptide containing e.g. the aminoacid sequence NH₂-GRKKRRQRRR—COOH (L-TAT) [SEQ ID NO: 5].

According to another more preferred embodiment the trafficking sequence(being included in the first domain of the chimeric peptide as usedherein) may comprise a D retro-inverso peptide of the sequences aspresented above, i.e. the D retro-inverso sequence of the generic TATsequence having the sequence NH₂—X_(n) ^(b)—RRRQRRKKR—X_(n) ^(b)—COOH(D-generic-TAT (s)) [SEQ ID NO: 8] and/or XXXXRRRQRRKKRXXXX(D-generic-TAT) [SEQ ID NO: 22]. Also here, X_(n) ^(b) is as definedabove (preferably representing D amino acids). Furthermore, the numberof “X_(n) ^(b)” residues in SEQ ID NOs:8 is not limited to the onedepicted, and may vary as described above. Most preferably, thetrafficking sequence as used herein may comprise the D retro-inversosequence NH₂—RRRQRRKKRG-COOH (D-TAT) [SEQ ID NO: 6].

According to another embodiment the trafficking sequence being includedin the first domain of the chimeric peptide as used herein may compriseor consist of variants of the trafficking sequences as defined above. A“variant of a trafficking sequence” is preferably a sequence derivedfrom a trafficking sequence as defined above, wherein the variantcomprises a modification, for example, addition, (internal) deletion(leading to fragments) and/or substitution of at least one amino acidpresent in the trafficking sequence as defined above. Such (a)modification(s) typically comprise(s) 1 to 20, preferably 1 to 10 andmore preferably 1 to 5 substitutions, additions and/or deletions ofamino acids. Furthermore, the variant preferably exhibits a sequenceidentity with the trafficking sequence as defined above, more preferablywith any of SEQ ID NOs: 5 to 8 or 21-22, of at least about 30%, 50%,70%, 80%, 90%, 95%, 98% or even 99%.

Preferably, such a modification of the trafficking sequence beingincluded in the first domain of the chimeric peptide as used hereinleads to a trafficking sequence with increased or decreased stability.Alternatively, variants of the trafficking sequence can be designed tomodulate intracellular localization of the chimeric peptide as usedherein. When added exogenously, such variants as defined above aretypically designed such that the ability of the trafficking sequence toenter cells is retained (i.e. the uptake of the variant of thetrafficking sequence into the cell is substantially similar to that ofthe native protein used a trafficking sequence). For example, alterationof the basic region thought to be important for nuclear localization(see e.g. Dang and Lee, J. Biol. Chem. 264: 18019-18023 (1989); Hauberet al., J. Virol. 63: 1181-1187 (1989); et al., J. Virol. 63: 1-8(1989)) can result in a cytoplasmic location or partially cytoplasmiclocation of the trafficking sequence, and therefore, of the JNKinhibitor sequence as component of the chimeric peptide as used herein.Additional to the above, further modifications may be introduced intothe variant, e.g. by linking e.g. cholesterol or other lipid moieties tothe trafficking sequence to produce a trafficking sequence havingincreased membrane solubility. Any of the above disclosed variants ofthe trafficking sequences being included in the first domain of thechimeric peptide as used herein can be produced using techniquestypically known to a skilled person (see e.g. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Molecular cloning: A laboratory manual. 2ndedition. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)

As a second domain the chimeric peptide as used herein typicallycomprises an JNK inhibitor sequence, selected from any of the JNKinhibitor sequences as defined above, including variants, fragmentsand/or derivatives of these JNK inhibitor sequences.

Both domains, i.e. the first and the second domain(s), of the chimericpeptide as used herein, may be linked such as to form a functional unit.Any method for linking the first and second domain(s) as generally knownin the art may be applied.

According to one embodiment, the first and the second domain(s) of thechimeric peptide as used herein are preferably linked by a covalentbond. A covalent bond, as defined herein, may be e.g. a peptide bond,which may be obtained by expressing the chimeric peptide as definedabove as a fusion protein. Fusion proteins, as described herein, can beformed and used in ways analogous to or readily adaptable from standardrecombinant DNA techniques, as described below. However, both domainsmay also be linked via side chains or may be linked by a chemical linkermoiety.

The first and/or second domains of the chimeric peptide as used hereinmay occur in one or more copies in said chimeric peptide. If bothdomains are present in a single copy, the first domain may be linkedeither to the N-terminal or the C-terminal end of the second domain. Ifpresent in multiple copies, the first and second domain(s) may bearranged in any possible order. E.g. the first domain can be present inthe chimeric peptide as used herein in a multiple copy number, e.g. intwo, three or more copies, which are preferably arranged in consecutiveorder. Then, the second domain may be present in a single copy occurringat the N- or C-terminus of the sequence comprising the first domain.Alternatively, the second domain may be present in a multiple copynumber, e.g. in two, three or more copies, and the first domain may bepresent in a single copy. According to both alternatives, first andsecond domain(s) can take any place in a consecutive arrangement.Exemplary arrangements are shown in the following: e.g. firstdomain—first domain—first domain—second domain; first domain—firstdomain—second domain—first domain; first domain—second domain—firstdomain—first domain; or e.g. second domain—first domain—firstdomain—first domain. It is well understood for a skilled person thatthese examples are for illustration purposes only and shall not limitthe scope of the invention thereto. Thus, the number of copies and thearrangement may be varied as defined initially.

Preferably, the first and second domain(s) may be directly linked witheach other without any linker. Alternatively, they may be linked witheach other via a linker sequence comprising 1 to 10, preferably 1 to 5amino acids. Amino acids forming the linker sequence are preferablyselected from glycine or proline as amino acid residues. Morepreferably, the first and second domain(s) may be separated by eachother by a hinge of two, three or more proline residues between thefirst and second domain(s).

The chimeric peptide as defined above and as used herein, comprising atleast one first and at least one second domain, may be composed ofL-amino acids, D-amino acids, or a combination of both. Therein, eachdomain (as well as the linkers used) may be composed of L-amino acids,D-amino acids, or a combination of both (e.g. D-TAT and L-IB1(s) orL-TAT and D-IB1(s), etc.). Preferably, the chimeric peptide as usedherein may comprise at least 1 or even 2, preferably at least 3, 4 or 5,more preferably at least 6, 7, 8 or 9 and even more preferably at least10 or more D- and/or L-amino acids, wherein the D- and/or L-amino acidsmay be arranged in the chimeric peptide as used herein in a blockwise, anon-blockwise or in an alternate manner.

According to a specific embodiment the chimeric peptide as used hereincomprises or consists of the L-amino acid chimeric peptides according tothe generic L-TAT-IB peptide NH₂—X_(n) ^(b)—RKKRRQRRR-X_(n) ^(b)—X_(n)^(a)—RPTTLXLXXXXXXXQD-X_(n) ^(b)—COOH (L-TAT-IB (generic) (s)) [SEQ IDNO: 10], wherein X, X_(n) ^(a) and X_(n) ^(b) are preferably as definedabove. More preferably, the chimeric peptide as used herein comprises orconsists of the L-amino acid chimeric peptideNH₂-GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQD-COOH (L-TAT-IB (s)) [SEQ ID NO: 9].Alternatively or additionally, the chimeric peptide as used hereincomprises or consists of the L-amino acid chimeric peptide sequenceGRKKRRQRRR PPDTYRPKRP TTLNLFPQVP RSQDT (L-TAT-IB1) [SEQ ID NO: 23], orXXXXXXXRKK RRQRRRXXXX XXXXRPTTLX LXXXXXXXQD S/TX (L-TAT-IB generic) [SEQID NO: 24], wherein X is preferably also as defined above, or thechimeric peptide as used herein comprises or consists of the L-aminoacid chimeric peptide sequence RKKRRQRRRPPRPKRPTTLNLFPQVPRSQD(L-TAT-IB1(s1)) [SEQ ID NO: 27], GRKKRRQRRRX_(n) ^(c)RPKRPTTLNLFPQVPRSQD(L-TAT-IB1(s2)) [SEQ ID NO: 28], or RKKRRQRRRX_(n)^(c)RPKRPTTLNLFPQVPRSQD (L-TAT-IB1(s3)) [SEQ ID NO: 29]. In thiscontext, each X typically represents an amino acid residue as definedabove, more preferably X_(n) ^(c) represents a contiguous stretch ofpeptide residues, each X independently selected from each other fromglycine or proline, e.g. a monotonic glycine stretch or a monotonicproline stretch, wherein n (the number of repetitions of X_(n) ^(c)) istypically 0-5, 5-10, 10-15, 15-20, 20-30 or even more, preferably 0-5 or5-10. X_(n) ^(c) may represent either D or L amino acids.

According to an alternative specific embodiment the chimeric peptide asused herein comprises or consists of D-amino acid chimeric peptides ofthe above disclosed L-amino acid chimeric peptides. Exemplary Dretro-inverso chimeric peptides according to the present invention aree.g. the generic D-TAT-IB peptide NH₂—X_(n) ^(b)-DQXXXXXXXLXLTTPR—X_(n)^(a)—X_(n) ^(b)—RRRQRRKKR—X_(n) ^(b)—COOH (D-TAT-IB (generic) (s)) [SEQID NO: 12]. Herein, X, X_(n) ^(a) and X_(n) ^(b) are preferably asdefined above (preferably representing D amino acids). More preferably,the chimeric peptide as used herein comprises or consists of D-aminoacid chimeric peptides according to the TAT-IB1 peptideNH₂-DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG-COOH (D-TAT-IB1(s)) [SEQ ID NO: 11].Alternatively or additionally, the chimeric peptide as used hereincomprises or consists of the D-amino acid chimeric peptide sequenceTDQSRPVQPFLNLTTPRKPRYTDPPRRRQRRKKRG (D-TAT-IB1) [SEQ ID NO: 25], orXT/SDQXXXXXXXLXLTTPRXXXXXXXXRRRQRRKKRXXXXXXX (D-TAT-IB generic) [SEQ IDNO: 26], wherein X is preferably also as defined above, or the chimericpeptide as used herein comprises or consists of the D-amino acidchimeric peptide sequence DQSRPVQPFLNLTTPRKPRPPRRRQRRKKR (D-TAT-IB1(s1))[SEQ ID NO: 30], DQSRPVQPFLNLTTPRKPRX_(n) ^(c)RRRQRRKKRG (D-TAT-IB1(s2))[SEQ ID NO: 31], or DQSRPVQPFLNLTTPRKPRX_(n) ^(c)RRQRRKKR(D-TAT-IB1(s3)) [SEQ ID NO: 32]. X_(n) ^(c) may be as defined above.

The first and second domain(s) of the chimeric peptide as defined abovemay be linked to each other by chemical or biochemical coupling carriedout in any suitable manner known in the art, e.g. by establishing apeptide bond between the first and the second domain(s) e.g. byexpressing the first and second domain(s) as a fusion protein, or e.g.by crosslinking the first and second domain(s) of the chimeric peptideas defined above.

Many known methods suitable for chemical crosslinking of the first andsecond domain(s) of the chimeric peptide as defined above arenon-specific, i.e. they do not direct the point of coupling to anyparticular site on the transport polypeptide or cargo macromolecule. Asa result, use of non-specific crosslinking agents may attack functionalsites or sterically block active sites, rendering the conjugatedproteins biologically inactive. Thus, preferably such crosslinkingmethods are used, which allow a more specific coupling of the first andsecond domain(s).

In this context, one way to increasing coupling specificity is a directchemical coupling to a functional group present only once or a few timesin one or both of the first and second domain(s) to be crosslinked. Forexample, cysteine, which is the only protein amino acid containing athiol group, occurs in many proteins only a few times. Also, forexample, if a polypeptide contains no lysine residues, a crosslinkingreagent specific for primary amines will be selective for the aminoterminus of that polypeptide. Successful utilization of this approach toincrease coupling specificity requires that the polypeptide have thesuitably rare and reactive residues in areas of the molecule that may bealtered without loss of the molecule's biological activity. Cysteineresidues may be replaced when they occur in parts of a polypeptidesequence where their participation in a crosslinking reaction wouldotherwise likely interfere with biological activity. When a cysteineresidue is replaced, it is typically desirable to minimize resultingchanges in polypeptide folding. Changes in polypeptide folding areminimized when the replacement is chemically and sterically similar tocysteine. For these reasons, serine is preferred as a replacement forcysteine. As demonstrated in the examples below, a cysteine residue maybe introduced into a polypeptide's amino acid sequence for crosslinkingpurposes. When a cysteine residue is introduced, introduction at or nearthe amino or carboxy terminus is preferred. Conventional methods areavailable for such amino acid sequence modifications, wherein thepolypeptide of interest is produced by chemical synthesis or viaexpression of recombinant DNA.

Coupling of the first and second domain(s) of the chimeric peptide asdefined above and used herein can also be accomplished via a coupling orconjugating agent. There are several intermolecular crosslinkingreagents which can be utilized (see for example, Means and Feeney,CHEMICAL MODIFICATION OF PROTEINS, Holden-Day, 1974, pp. 39-43). Amongthese reagents are, for example, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N,N′-(1,3-phenylene) bismaleimide (both of whichare highly specific for sulfhydryl groups and form irreversiblelinkages); N, N′-ethylene-bis-(iodoacetamide) or other such reagenthaving 6 to 11 carbon methylene bridges (which are relatively specificfor sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which formsirreversible linkages with amino and tyrosine groups). Othercrosslinking reagents useful for this purpose include: p,p′-difluoro-m,m′-dinitrodiphenylsulfone which forms irreversible crosslinkages withamino and phenolic groups); dimethyl adipimidate (which is specific foramino groups); phenol-1,4 disulfonylchloride (which reacts principallywith amino groups); hexamethylenediisocyanate or diisothiocyanate, orazophenyl-p-diisocyanate (which reacts principally with amino groups);glutaraldehyde (which reacts with several different side chains) anddisdiazobenzidine (which reacts primarily with tyrosine and histidine).

Crosslinking reagents used for crosslinking the first and seconddomain(s) of the chimeric peptide as defined above may behomobifunctional, i.e. having two functional groups that undergo thesame reaction. A preferred homobifunctional crosslinking reagent isbismaleimidohexane (“BMH”). BMH contains two maleimide functionalgroups, which react specifically with sulfhydryl-containing compoundsunder mild conditions (pH 6.5-7.7). The two maleimide groups areconnected by a hydrocarbon chain. Therefore, BMH is useful forirreversible crosslinking of polypeptides that contain cysteineresidues.

Crosslinking reagents used for crosslinking the first and seconddomain(s) of the chimeric peptide as defined above may also beheterobifunctional. Heterobifunctional crosslinking agents have twodifferent functional groups, for example an amine-reactive group and athiol-reactive group, that will crosslink two proteins having freeamines and thiols, respectively. Examples of heterobifunctionalcrosslinking agents are succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (“SMCC”),m-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), and succinimide4-(p-maleimidophenyl)butyrate (“SMPB”), an extended chain analog of MBS.The succinimidyl group of these crosslinkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue.

Crosslinking reagents suitable for crosslinking the first and seconddomain(s) of the chimeric peptide as defined above often have lowsolubility in water. A hydrophilic moiety, such as a sulfonate group,may thus be added to the crosslinking reagent to improve its watersolubility. In this respect, Sulfo-MBS and Sulfo-SMCC are examples ofcrosslinking reagents modified for water solubility, which may be usedaccording to the present invention.

Likewise, many crosslinking reagents yield a conjugate that isessentially non-cleavable under cellular conditions. However, somecrosslinking reagents particularly suitable for crosslinking the firstand second domain(s) of the chimeric peptide as defined above contain acovalent bond, such as a disulfide, that is cleavable under cellularconditions. For example, Traut's reagent,dithiobis(succinimidylpropionate) (“DSP”), and N-succinimidyl3-(2-pyridyldithio)propionate (“SPDP”) are well-known cleavablecrosslinkers. The use of a cleavable crosslinking reagent permits thecargo moiety to separate from the transport polypeptide after deliveryinto the target cell. Direct disulfide linkage may also be useful.

Numerous crosslinking reagents, including the ones discussed above, arecommercially available. Detailed instructions for their use are readilyavailable from the commercial suppliers. A general reference on proteincrosslinking and conjugate preparation is: Wong, CHEMISTRY OF PROTEINCONJUGATION AND CROSSLINKING, CRC Press (1991).

Chemical crosslinking of the first and second domain(s) of the chimericpeptide as defined above may include the use of spacer arms. Spacer armsprovide intramolecular flexibility or adjust intramolecular distancesbetween conjugated moieties and thereby may help preserve biologicalactivity. A spacer arm may be in the form of a polypeptide moiety thatincludes spacer amino acids, e.g. proline. Alternatively, a spacer armmay be part of the crosslinking reagent, such as in “long-chain SPDP”(Pierce Chem. Co., Rockford, Ill., cat. No. 21651 H).

Furthermore, variants, fragments or derivatives of one of the abovedisclosed chimeric peptides may be used herein. With regard to fragmentsand variants it is generally referred to the definition given above forJNK inhibitor sequences.

Particularly, in the context of the present invention, a “variant of achimeric peptide” is preferably a sequence derived from any of thesequences according to SEQ ID NOs: 9 to 12 and 23 to 32, wherein thechimeric variant comprises amino acid alterations of the chimericpeptides according to SEQ ID NOs: 9 to 12 and 23 to 32 as used herein.Such alterations typically comprise 1 to 20, preferably 1 to 10 and morepreferably 1 to 5 substitutions, additions and/or deletions (leading tofragments) of amino acids according to SEQ ID NOs: 9 to 12 and 23 to 32,wherein the altered chimeric peptide as used herein exhibits a sequenceidentity with any of the sequences according to SEQ ID NOs: 9-12 and 23to 32 of at least about 30%, 50%, 70%, 80%, or 95%, 98%, or even 99%.Preferably, these variants retain the biological activity of the firstand the second domain as contained in the chimeric peptide as usedherein, i.e. the trafficking activity of the first domain as disclosedabove and the activity of the second domain for binding JNK and/orinhibiting the activation of at least one JNK activated transcriptionfactor.

Accordingly, the chimeric peptide as used herein also comprisesfragments of the afore disclosed chimeric peptides, particularly of thechimeric peptide sequences according to any of SEQ ID NOs: 9 to 12 and23 to 32. Thus, in the context of the present invention, a “fragment ofthe chimeric peptide” is preferably a sequence derived any of thesequences according to SEQ ID NOs: 9 to 12 and 23 to 32, wherein thefragment comprises at least 4 contiguous amino acids of any of SEQ IDNOs: 9 to 12 and 23 to 32. This fragment preferably comprises a lengthwhich is sufficient to allow specific recognition of an epitope from anyof these sequences and to transport the sequence into the cells, thenucleus or a further preferred location. Even more preferably, thefragment comprises 4 to 18, 4 to 15, or most preferably 4 to 10contiguous amino acids of any of SEQ ID NOs: 9 to 12 and 23 to 32.Fragments of the chimeric peptide as used herein further may be definedas a sequence sharing a sequence identity with any of the sequencesaccording to any of SEQ ID NOs: 99 to 12 and 23 to 32 of at least about30%, 50%, 70%, 80%, or 95%, 98%, or even 99%.

Finally, the chimeric peptide as used herein also comprises derivativesof the afore disclosed chimeric peptides, particularly of the chimericpeptide sequences according to any of SEQ ID NOs: 9 to 12 and 23 to 32.

A particularly preferred use of the present invention is the use of aJNK inhibitor (poly-)peptide consisting of or comprising the amino acidsequence of SEQ ID NO: 11, or consisting of or comprising an amino acidsequence sharing a sequence identity of at least about 30%, 50%, 70%,80%, 90%, 92% or even 95% with SEQ ID NO: 11, for the treatment ofinflammatory eye diseases, in particular for the treatment of uveitis,for example for the treatment of anterior uveitis, intermediate uveitis,posterior uveitis or panuveitis. The JNK inhibitor (poly-)peptideconsisting of or comprising the amino acid sequence of SEQ ID NO: 11, orconsisting of or comprising an amino acid sequence sharing a sequenceidentity of at least about 30%, 50%, 70%, 80%, 90%, 92% or even 95% withSEQ ID NO: 11 may be administered for example locally to the eye orsystemically. However, the present application also clearly contemplatesthe use of other JNK inhibitor chimeric (poly-)peptides, i.e. where theJNK inhibitor poly-)peptide used does not consist of or comprise theamino acid sequence of SEQ ID NO: 11 for the treatment of inflammatoryeye diseases, in particular for the treatment of uveitis, for examplefor the treatment of anterior uveitis, intermediate uveitis, posterioruveitis or panuveitis.

Furthermore, the inventors also clearly contemplate the use of the JNKinhibitor (poly-)peptides of the present invention, in particular wherethe JNK inhibitor poly-)peptide used consists of or comprises the aminoacid sequence of SEQ ID NO: 11 or consists of or comprises an amino acidsequence sharing a sequence identity of at least about 30%, 50%, 70%,80%, 90%, 92% or even 95% with SEQ ID NO: 11, for the treatment ofinflammatory eye diseases other than inflammation of the uvea and/orretina, e.g. for the treatment of inflammatory eye diseases which arenot uveitis and/or retinitis. Moreover, it must be noted that thepresent invention does in particular not contemplate the treatment of(non-inflammatory) retinopathy.

The present invention additionally refers to the use of nucleic acidsequences encoding JNK inhibitor sequences as defined above, chimericpeptides or their fragments, variants or derivatives, all as definedabove, for the preparation of a pharmaceutical composition for treatingnon-chronic or chronic inflammatory eye diseases in a subject as definedherein. A preferable suitable nucleic acid encoding an JNK inhibitorsequence as used herein is typically chosen from human IB1 nucleic acid(GenBank Accession No. (AF074091), rat IB1 nucleic acid (GenBankAccession No. AF 108959), or human IB2 (GenBank Accession No AF218778)or from any nucleic acid sequence encoding any of the sequences asdefined above, i.e. any sequence according to SEQ ID NO: 1-26.

Nucleic acids encoding the JNK inhibitor sequences as used herein orchimeric peptides as used herein may be obtained by any method known inthe art (e.g. by PCR amplification using synthetic primers hybridizableto the 3′- and 5′-termini of the sequence and/or by cloning from a cDNAor genomic library using an oligonucleotide sequence specific for thegiven gene sequence).

Additionally, nucleic acid sequences are disclosed herein as well, whichhybridize under stringent conditions with the appropriate strand codingfor a (native) JNK inhibitor sequence or chimeric peptide as definedabove. Preferably, such nucleic acid sequences comprise at least 6(contiguous) nucleic acids, which have a length sufficient to allow forspecific hybridization. More preferably, such nucleic acid sequencescomprise 6 to 38, even more preferably 6 to 30, and most preferably 6 to20 or 6 to 10 (contiguous) nucleic acids.

“Stringent conditions” are sequence dependent and will be differentunder different circumstances. Generally, stringent conditions can beselected to be about 5° C. lower than the thermal melting point (TM) forthe specific sequence at a defined ionic strength and pH. The TM is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration is atleast about 0.02 molar at pH 7 and the temperature is at least about 60°C. As other factors may affect the stringency of hybridization(including, among others, base composition and size of the complementarystrands), the presence of organic solvents and the extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one.

“High stringency conditions” may comprise the following, e.g. Step 1:Filters containing DNA are pretreated for 8 hours to overnight at 65° C.in buffer composed of 6*SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02%PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA.Step 2: Filters are hybridized for 48 hours at 65° C. in the aboveprehybridization mixture to which is added 100 mg/ml denatured salmonsperm DNA and 5-20*10⁶ cpm of ³²P-labeled probe. Step 3: Filters arewashed for 1 hour at 37° C. in a solution containing 2*SSC, 0.01% PVP,0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1*SSC at50° C. for 45 minutes. Step 4: Filters are autoradiographed. Otherconditions of high stringency that may be used are well known in the art(see e.g. Ausubel et al., (eds.), 1993, Current Protocols in MolecularBiology, John Wiley and Sons, NY; and Kriegler, 1990, Gene Transfer andExpression, a Laboratory Manual, Stockton Press, NY).

“Moderate stringency conditions” can include the following: Step 1:Filters containing DNA are pretreated for 6 hours at 55° C. in asolution containing 6*SSC, 5*Denhardt's solution, 0.5% SDS and 100 mg/mldenatured salmon sperm DNA. Step 2: Filters are hybridized for 18-20hours at 55° C. in the same solution with 5-20*10⁶ cpm ³²P-labeled probeadded. Step 3: Filters are washed at 37° C. for 1 hour in a solutioncontaining 2*SSC, 0.1% SDS, then washed twice for 30 minutes at 60° C.in a solution containing 1*SSC and 0.1% SDS. Step 4: Filters are blotteddry and exposed for autoradiography. Other conditions of moderatestringency that may be used are well-known in the art (see e.g. Ausubelet al., (eds.), 1993, Current Protocols in Molecular Biology, John Wileyand Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, aLaboratory Manual, Stockton Press, NY).

Finally, “low stringency conditions” can include: Step 1: Filterscontaining DNA are pretreated for 6 hours at 40° C. in a solutioncontaining 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA,0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA.Step 2: Filters are hybridized for 18-20 hours at 40° C. in the samesolution with the addition of 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106cpm^(S2)P-labeled probe. Step 3: Filters are washed for 1.5 hours at 55C in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA,and 0.1% SDS. The wash solution is replaced with fresh solution andincubated an additional 1.5 hours at 60° C. Step 4: Filters are blotteddry and exposed for autoradiography. If necessary, filters are washedfor a third time at 65-68° C. and reexposed to film. Other conditions oflow stringency that may be used are well known in the art (e.g. asemployed for cross-species hybridizations). See e.g. Ausubel et al.,(eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley andSons, NY; and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORYMANUAL, Stockton Press, NY.

The nucleic acid sequences as defined above according to the presentinvention can be used to express peptides, i.e. an JNK inhibitorsequence as used herein or an chimeric peptide as used herein foranalysis, characterization or therapeutic use; as markers for tissues inwhich the corresponding peptides (as used herein) are preferentiallyexpressed (either constitutively or at a particular stage of tissuedifferentiation or development or in disease states). Other uses forthese nucleic acids include, e.g. molecular weight markers in gelelectrophoresis-based analysis of nucleic acids.

According to a further embodiment of the present invention, expressionvectors may be used for the above purposes for recombinant expression ofone or more JNK inhibitor sequences and/or chimeric peptides as definedabove. The term “expression vector” is used herein to designate eithercircular or linear DNA or RNA, which is either double-stranded orsingle-stranded. It further comprises at least one nucleic acid asdefined above to be transferred into a host cell or into a unicellularor multicellular host organism. The expression vector as used hereinpreferably comprises a nucleic acid as defined above encoding the JNKinhibitor sequence as used herein or a fragment or a variant thereof, orthe chimeric peptide as used herein, or a fragment or a variant thereof.Additionally, an expression vector according to the present inventionpreferably comprises appropriate elements for supporting expressionincluding various regulatory elements, such as enhancers/promoters fromviral, bacterial, plant, mammalian, and other eukaryotic sources thatdrive expression of the inserted polynucleotide in host cells, such asinsulators, boundary elements, LCRs (e.g. described by Blackwood andKadonaga (1998), Science 281, 61-63) or matrix/scaffold attachmentregions (e.g. described by Li, Harju and Peterson, (1999), Trends Genet.15, 403-408). In some embodiments, the regulatory elements areheterologous (i.e. not the native gene promoter). Alternately, thenecessary transcriptional and translational signals may also be suppliedby the native promoter for the genes and/or their flanking regions.

The term “promoter” as used herein refers to a region of DNA thatfunctions to control the transcription of one or more nucleic acidsequences as defined above, and that is structurally identified by thepresence of a binding site for DNA-dependent RNA-polymerase and of otherDNA sequences, which interact to regulate promoter function. Afunctional expression promoting fragment of a promoter is a shortened ortruncated promoter sequence retaining the activity as a promoter.Promoter activity may be measured by any assay known in the art (seee.g. Wood, de Wet, Dewji, and DeLuca, (1984), Biochem Biophys. Res.Commun. 124, 592-596; Seliger and McElroy, (1960), Arch. Biochem.Biophys. 88, 136-141) or commercially available from Promega®).

An “enhancer region” to be used in the expression vector as definedherein, typically refers to a region of DNA that functions to increasethe transcription of one or more genes. More specifically, the term“enhancer”, as used herein, is a DNA regulatory element that enhances,augments, improves, or ameliorates expression of a gene irrespective ofits location and orientation vis-á-vis the gene to be expressed, and maybe enhancing, augmenting, improving, or ameliorating expression of morethan one promoter.

The promoter/enhancer sequences to be used in the expression vector asdefined herein, may utilize plant, animal, insect, or fungus regulatorysequences. For example, promoter/enhancer elements can be used fromyeast and other fungi (e.g. the GAL4 promoter, the alcohol dehydrogenasepromoter, the phosphoglycerol kinase promoter, the alkaline phosphatasepromoter). Alternatively, or in addition, they may include animaltranscriptional control regions, e.g. (i) the insulin gene controlregion active within pancreatic beta-cells (see e.g. Hanahan, et al.,1985. Nature 315: 115-122); (ii) the immunoglobulin gene control regionactive within lymphoid cells (see e.g. Grosschedl, et al., 1984, Cell38: 647-658); (iii) the albumin gene control region active within liver(see e.g. Pinckert, et al., 1987. Genes and Dev 1: 268-276; (iv) themyelin basic protein gene control region active within brainoligodendrocyte cells (see e.g. Readhead, et al., 1987, Cell 48:703-712); and (v) the gonadotropin-releasing hormone gene control regionactive within the hypothalamus (see e.g. Mason, et al., 1986, Science234: 1372-1378), and the like.

Additionally, the expression vector as defined herein may comprise anamplification marker. This amplification marker may be selected from thegroup consisting of, e.g. adenosine deaminase (ADA), dihydrofolatereductase (DHFR), multiple drug resistance gene (MDR), ornithinedecarboxylase (ODC) and N-(phosphonacetyl)-L-aspartate resistance (CAD).

Exemplary expression vectors or their derivatives suitable for thepresent invention particularly include, e.g. human or animal viruses(e.g. vaccinia virus or adenovirus); insect viruses (e.g. baculovirus);yeast vectors; bacteriophage vectors (e.g. lambda phage); plasmidvectors and cosmid vectors.

The present invention additionally may utilize a variety of host-vectorsystems, which are capable of expressing the peptide coding sequence(s)of nucleic acids as defined above. These include, but are not limitedto: (i) mammalian cell systems that are infected with vaccinia virus,adenovirus, and the like; (ii) insect cell systems infected withbaculovirus and the like; (iii) yeast containing yeast vectors or (iv)bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmidDNA. Depending upon the host-vector system utilized, any one of a numberof suitable transcription and translation elements may be used.

Preferably, a host cell strain, suitable for such a host-vector system,may be selected that modulates the expression of inserted sequences ofinterest, or modifies or processes expressed peptides encoded by thesequences in the specific manner desired. In addition, expression fromcertain promoters may be enhanced in the presence of certain inducers ina selected host strain; thus facilitating control of the expression of agenetically-engineered peptide. Moreover, different host cells possesscharacteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g. glycosylation,phosphorylation, and the like) of expressed peptides. Appropriate celllines or host systems may thus be chosen to ensure the desiredmodification and processing of the foreign peptide is achieved. Forexample, peptide expression withina bacterial system can be used toproduce an non-glycosylated core peptide; whereas expression withinmammalian cells ensures “native” glycosylation of a heterologouspeptide.

The present invention further provides the use of antibodies directedagainst the JNK inhibitor sequences and/or chimeric peptides asdescribed above, for preparing a pharmaceutical composition for thetreatment of non-chronic or chronic inflammatory eye diseases as definedherein. Furthermore, efficient means for production of antibodiesspecific for JNK inhibitor sequences according to the present invention,or for chimeric peptides containing such an inhibitor sequence, aredescribed and may be utilized for this purpose.

According to the invention, JNK inhibitor sequences and/or chimericpeptides as defined herein, as well as, fragments, variants orderivatives thereof, may be utilized as immunogens to generateantibodies that immunospecifically bind these peptide components. Suchantibodies include, e.g. polyclonal, monoclonal, chimeric, single chain,Fab fragments and a Fab expression library. In a specific embodiment thepresent invention provides antibodies to chimeric peptides or to JNKinhibitor sequences as defined above. Various procedures known withinthe art may be used for the production of these antibodies.

By way of example, various host animals may be immunized for productionof polyclonal antibodies by injection with any chimeric peptide or JNKinhibitor sequence as defined above. Various adjuvants may be usedthereby to increase the immunological response which include, but arenot limited to, Freund's (complete and incomplete) adjuvant, mineralgels (e.g. aluminum hydroxide), surface active substances (e.g.lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,dinitrophenol, etc.), CpG, polymers, Pluronics, and human adjuvants suchas Bacille Calmette-Guerin and Corynebacterium parvum.

For preparation of monoclonal antibodies directed towards a chimericpeptide or a JNK inhibitor sequence as defined above, any technique maybe utilized that provides for the production of antibody molecules bycontinuous cell line culture. Such techniques include, but are notlimited to, the hybridoma technique (see Kohler and Milstein, 1975.Nature 256: 495-497); the trioma technique; the human B-cell hybridomatechnique (see Kozbor, et al., 1983, Immunol Today 4: 72) and the EBVhybridoma technique to produce human monoclonal antibodies (see Cole, etal., 1985. In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). Human monoclonal antibodies may be utilized in thepractice of the present invention and may be produced by the use ofhuman hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985. In: Monoclonal Antibodies and CancerTherapy (Alan R. Liss, Inc., pp. 77-96).

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to the JNK inhibitor sequencesand/or chimeric peptides (see e.g. U.S. Pat. No. 4,946,778) as definedherein. In addition, methods can be adapted for the construction of Fabexpression libraries (see e.g. Huse et al., 1989. Science 246:1275-1281) to allow rapid and effective identification of monoclonal Fabfragments with the desired specificity for these JNK inhibitor sequencesand/or chimeric peptides. Non-human antibodies can be “humanized” bytechniques well known in the art (see e.g. U.S. Pat. No. 5,225,539).Antibody fragments that contain the idiotypes to a JNK inhibitorsequences and/or chimeric peptide as defined herein may be produced bytechniques known in the art including, e.g. (i) a F(ab′)₂ fragmentproduced by pepsin digestion of an antibody molecule; (ii) a Fabfragment generated by reducing the disulfide bridges of an F(ab′),fragment; (iii) a Fab fragment generated by the treatment of theantibody molecule with papain and a reducing agent and (iv) Fvfragments.

In one embodiment of this invention, methods, that may be utilized forthe screening of antibodies and which possess the desired specificityinclude, but are not limited to, enzyme-linked immunosorbent assay(ELISA) and other immunologically-mediated techniques known within theart. In a specific embodiment, selection of antibodies that are specificto a particular epitope of an JNK inhibitor sequence and/or an chimericpeptide as defined herein (e.g. a fragment thereof typically comprisinga length of from 5 to 20, preferably 8 to 18 and most preferably 8 to 11amino acids) is facilitated by generation of hybridomas that bind to thefragment of an JNK inhibitor sequence and/or an chimeric peptide, asdefined herein, possessing such an epitope. These antibodies that arespecific for an epitope as defined above are also provided herein.

The antibodies as defined herein may be used in methods known within theart referring to the localization and/or quantification of an JNKinhibitor sequence (and/or correspondingly to a chimeric peptide asdefined above), e.g. for use in measuring levels of the peptide withinappropriate physiological samples, for use in diagnostic methods, or foruse in imaging the peptide, and the like.

The JNK inhibitor sequences, chimeric peptides, nucleic acids, vectors,host cells and/or antibodies as defined according to the invention canbe formulated in a pharmaceutical composition, which may be applied inthe prevention or treatment of any of the diseases as defined herein,particularly in the prevention or treatment of non-chronic or chronicinflammatory eye diseases as defined herein. Typically, such apharmaceutical composition used according to the present inventionincludes as an active component, e.g.: (i) any one or more of the JNKinhibitor sequences and/or chimeric peptides as defined above, and/orvariants, fragments or derivatives thereof, particularly JNK inhibitorsequences according to any of sequences of SEQ ID NOs: 1 to 4 and 13 to20 and 33-100 and/or chimeric peptides according to any of sequences ofSEQ ID NOs: 9 to 12 and 23 to 32, and/or JNK inhibitor sequencesaccording to any of sequences of SEQ ID NOs: 1 to 4 and 13 to 20 and33-100 comprising a trafficking sequence according to any of SEQ ID NOs:5 to 8 and 21 to 22, or variants or fragments thereof within the abovedefinitions; and/or (ii) nucleic acids encoding an JNK inhibitorsequence and/or an chimeric peptide as defined above and/or variants orfragments thereof, and/or (iii) cells comprising any one or more of theJNK inhibitor sequences and/or chimeric peptides, and/or variants,fragments or derivatives thereof, as defined above and/or (iv) cellstransfected with a vector and/or nucleic acids encoding an JNK inhibitorsequence and/or an chimeric peptide as defined above and/or variants orfragments thereof.

According to a preferred embodiment, such a pharmaceutical compositionas used according to the present invention typically comprises a safeand effective amount of a component as defined above, preferably of atleast one JNK inhibitor sequence according to any of sequences of SEQ IDNOs: 1 to 4 and 13 to 20 and 33-100 and/or at least one chimeric peptideaccording to any of sequences of SEQ ID NOs: 9 to 12 and 23 to 32,and/or at least one JNK inhibitor sequence according to any of sequencesof SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 comprising a traffickingsequence according to any of SEQ ID NOs: 5-8 and 21 to 22, or variantsor fragments thereof within the above definitions, or at least onenucleic acids encoding same, or at least one vector, host cell orantibody as defined above.

The amount of a JNK-inhibitor sequence and chimeric peptide,respectively, in the pharmaceutical composition to be administered to asubject, may—without being limited thereto—have a very low dose. Thus,the dose may be much lower than for peptide drugs known in the art, suchas DTS-108 (Florence Meyer-Losic et al., Clin Cancer Res., 2008,2145-53). This has several positive aspects, for example a reduction ofpotential side reactions and a reduction in costs.

Preferably, the dose (per kg bodyweight) is in the range of up to 10mmol/kg, preferably up to 1 mmol/kg, more preferably up to 100 μmol/kg,even more preferably up to 10 μmol/kg, even more preferably up to 1μmol/kg, even more preferably up to 100 nmol/kg, most preferably up to50 nmol/kg.

Thus, the dose range may preferably be from about 1 pmol/kg to about 1mmol/kg, from about 10 pmol/kg to about 0.1 mmol/kg, from about 10pmol/kg to about 0.01 mmol/kg, from about 50 pmol/kg to about 1 μmol/kg,from about 100 pmol/kg to about 500 nmol/kg, from about 200 pmol/kg toabout 300 nmol/kg, from about 300 pmol/kg to about 100 nmol/kg, fromabout 500 pmol/kg to about 50 nmol/kg, from about 750 pmol/kg to about30 nmol/kg, from about 250 pmol/kg to about 5 nmol/kg, from about 1nmol/kg to about 10 nmol/kg, or a combination of any two of said values.

In this context, prescription of treatment, e.g. decisions on dosageetc. when using the above pharmaceutical composition is typically withinthe responsibility of general practitioners and other medical doctors,and typically takes account of the disorder to be treated, the conditionof the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in REMINGTON'SPHARMACEUTICAL SCIENCES, 16th edition, Osol, A. (ed), 1980. Accordingly,a “safe and effective amount” as defined above for components of thepharmaceutical compositions as used according to the present inventionmeans an amount of each or all of these components, that is sufficientto significantly induce a positive modification of a non-chronic orchronic inflammatory eye diseases as defined herein. At the same time,however, a “safe and effective amount” is small enough to avoid seriousside-effects, that is to say to permit a sensible relationship betweenadvantage and risk. The determination of these limits typically lieswithin the scope of sensible medical judgment. A “safe and effectiveamount” of such a component will vary in connection with the particularcondition to be treated and also with the age and physical condition ofthe patient to be treated, the severity of the condition, the durationof the treatment, the nature of the accompanying therapy, of theparticular pharmaceutically acceptable carrier used, and similarfactors, within the knowledge and experience of the accompanying doctor.The pharmaceutical compositions according to the invention can be usedaccording to the invention for human and also for veterinary medicalpurposes.

The pharmaceutical composition as used according to the presentinvention may furthermore comprise, in addition to one of thesesubstances, a (compatible) pharmaceutically acceptable carrier,excipient, buffer, stabilizer or other materials well known to thoseskilled in the art.

In this context, the expression “(compatible) pharmaceuticallyacceptable carrier” preferably includes the liquid or non-liquid basisof the composition. The term “compatible” means that the constituents ofthe pharmaceutical composition as used herein are capable of being mixedwith the pharmaceutically active component as defined above and with oneanother component in such a manner that no interaction occurs whichwould substantially reduce the pharmaceutical effectiveness of thecomposition under usual use conditions. Pharmaceutically acceptablecarriers must, of course, have sufficiently high purity and sufficientlylow toxicity to make them suitable for administration to a person to betreated.

If the pharmaceutical composition as used herein is provided in liquidform, the pharmaceutically acceptable carrier will typically compriseone or more (compatible) pharmaceutically acceptable liquid carriers.The composition may comprise as (compatible) pharmaceutically acceptableliquid carriers e.g. pyrogen-free water; isotonic saline or buffered(aqueous) solutions, e.g. phosphate, citrate etc. buffered solutions,vegetable oils, such as, for example, groundnut oil, cottonseed oil,sesame oil, olive oil, corn oil and oil from theobroma; polyols, suchas, for example, polypropylene glycol, glycerol, sorbitol, mannitol andpolyethylene glycol; alginic acid, etc. Particularly for injection ofthe pharmaceutical composition as used herein, a buffer, preferably anaqueous buffer, may be used.

If the pharmaceutical composition as used herein is provided in solidform, the pharmaceutically acceptable carrier will typically compriseone or more (compatible) pharmaceutically acceptable solid carriers. Thecomposition may comprise as (compatible) pharmaceutically acceptablesolid carriers e.g. one or more compatible solid or liquid fillers ordiluents or encapsulating compounds may be used as well, which aresuitable for administration to a person. Some examples of such(compatible) pharmaceutically acceptable solid carriers are e.g. sugars,such as, for example, lactose, glucose and sucrose; starches, such as,for example, corn starch or potato starch; cellulose and itsderivatives, such as, for example, sodium carboxymethylcellulose,ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin;tallow; solid glidants, such as, for example, stearic acid, magnesiumstearate; calcium sulphate, etc.

The precise nature of the (compatible) pharmaceutically acceptablecarrier or other material may depend on the route of administration. Thechoice of a (compatible) pharmaceutically acceptable carrier may thus bedetermined in principle by the manner in which the pharmaceuticalcomposition as used according to the invention is administered. Thepharmaceutical composition as used according to the invention can beadministered, for example, systemically. Routes for administrationinclude, for example, parenteral routes (e.g. via injection), such asintravenous, intramuscular, subcutaneous, intradermal, or transdermalroutes, etc., enteral routes, such as oral, or rectal routes, etc.,topical routes, such as nasal, or intranasal routes, etc., or otherroutes, such as epidermal routes or patch delivery. Partiularlypreferred is also the local administration at/in the eye, e.g.intravitreous administration, subconjuntival administration and/orinstillation.

The suitable amount of the pharmaceutical composition to be used can bedetermined by routine experiments with animal models. Such modelsinclude, without implying any limitation, rabbit, sheep, mouse, rat, dogand non-human primate models. Preferred unit dose forms for injectioninclude sterile solutions of water, physiological saline or mixturesthereof. The pH of such solutions should be adjusted to about 7.4.Suitable carriers for injection include hydrogels, devices forcontrolled or delayed release, polylactic acid and collagen matrices.Suitable pharmaceutically acceptable carriers for topical applicationinclude those, which are suitable for use in lotions, creams, gels andthe like. If the compound is to be administered perorally, tablets,capsules and the like are the preferred unit dose form. Thepharmaceutically acceptable carriers for the preparation of unit doseforms, which can be used for oral administration are well known in theprior art. The choice thereof will depend on secondary considerationssuch as taste, costs and storability, which are not critical for thepurposes of the present invention, and can be made without difficulty bya person skilled in the art.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carrier asdefined above, such as gelatin, and optionally an adjuvant. Liquidpharmaceutical compositions for oral administration generally mayinclude a liquid carrier as defined above, such as water, petroleum,animal or vegetable oils, mineral oil or synthetic oil. Physiologicalsaline solution, dextrose or other saccharide solution or glycols suchas ethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilizers, buffers,antioxidants and/or other additives may be included, as required.Whether it is a polypeptide, peptide, or nucleic acid molecule, otherpharmaceutically useful compound according to the present invention thatis to be given to an individual, administration is preferably in a“prophylactically effective amount or a “therapeutically effectiveamount” (as the case may be), this being sufficient to show benefit tothe individual. The actual amount administered, and rate and time-courseof administration, will depend on the nature and severity of what isbeing treated.

Prevention and/or treatment of a disease as defined herein typicallyincludes administration of a pharmaceutical composition as definedabove. The term “modulate” includes the suppression of expression of JNKwhen it is over-expressed in any of the above diseases. It alsoincludes, without being limited thereto, suppression of phosphorylationof c-jun, ATF2 or NFAT4 in any of the above diseases, for example, byusing at least one JNK inhibitor sequence according to any of sequencesof SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 and/or at least onechimeric peptide according to any of sequences of SEQ ID NOs: 9 to 12and 23 to 32, and/or at least one JNK inhibitor sequence according toany of sequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100comprising a trafficking sequence according to any of SEQ ID NOs: 5 to 8and 21 to 22, or variants or fragments thereof within the abovedefinitions, as a competitive inhibitor of the natural c-jun, ATF2 andNFAT4 binding site in a cell. The term “modulate” also includessuppression of hetero- and homomeric complexes of transcription factorsmade up of, without being limited thereto, c-jun, ATF2, or NFAT4 andtheir related partners, such as for example the AP-1 complex that ismade up of c-jun, AFT2 and c-fos. When a non-chronic or chronicinflammatory eye disease is associated with JNK overexpression, suchsuppressive JNK inhibitor sequences can be introduced to a cell. In someinstances, “modulate” may then include the increase of JNK expression,for example by use of an IB peptide-specific antibody that blocks thebinding of an IB-peptide to JNK, thus preventing JNK inhibition by theIB-related peptide.

Prevention and/or treatment of a subject with the pharmaceuticalcomposition as disclosed above may be typically accomplished byadministering (in vivo) an (“therapeutically effective”) amount of saidpharmaceutical composition to a subject, wherein the subject may be e.g.any mammal, e.g. a human, a primate, mouse, rat, dog, cat, cow, horse orpig. The term “therapeutically effective” means that the activecomponent of the pharmaceutical composition is of sufficient quantity toameliorate the non-chronic or chronic inflammatory eye disease.

Accordingly, peptides as defined above, e.g. at least one JNK inhibitorsequence according to any of sequences of SEQ ID NOs: 1 to 4 and 13 to20 and 33-100 and/or at least one chimeric peptide according to any ofsequences of SEQ ID NOs: 9 to 12 and 23 to 32, and/or at least one JNKinhibitor sequence according to any of sequences of SEQ ID NOs: 1 to 4and 13 to 20 and 33-100 comprising a trafficking sequence according toany of SEQ ID NOs: 5 to 8 and 21 to 22, or variants or fragments thereofwithin the above definitions, may be utilized in a specific embodimentof the present invention to treat non-chronic or chronic inflammatoryeye diseases.

Peptides as defined above and as contained in the inventivepharmaceutical composition may be also encoded by nucleic acids. This isparticularly advantageous, if the above peptides are administered forthe purpose of gene therapy. In this context, gene therapy refers totherapy that is performed by administration of a specific nucleic acidas defined above to a subject, e.g. by way of a pharmaceuticalcomposition as defined above, wherein the nucleic acid(s) exclusivelycomprise(s) L-amino acids. In this embodiment of the present invention,the nucleic acid produces its encoded peptide(s), which then serve(s) toexert a therapeutic effect by modulating function of the disease ordisorder. Any of the methods relating to gene therapy available withinthe art may be used in the practice of the present invention (see e.g.Goldspiel, et al., 1993. Clin Pharm 12: 488-505).

In a preferred embodiment, the nucleic acid as defined above and as usedfor gene therapy is part of an expression vector encoding and expressingany one or more of the IB-related peptides as defined above within asuitable host, i.e. an JNK inhibitor sequence according to any ofsequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 and/or achimeric peptide according to any of sequences of SEQ ID NOs: 9 to 12and 23 to 32, and/or an JNK inhibitor sequence according to any ofsequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 comprising atrafficking sequence according to any of SEQ ID NOs: 5 to 8 and 21 to22, or variants or fragments thereof within the above definitions. In aspecific embodiment, such an expression vector possesses a promoter thatis operably-linked to coding region(s) of a JNK inhibitor sequence. Thepromoter may be defined as above, e.g. inducible or constitutive, and,optionally, tissue-specific.

In another specific embodiment, a nucleic acid molecule as defined aboveis used for gene therapy, in which the coding sequences of the nucleicacid molecule (and any other desired sequences thereof) as defined aboveare flanked by regions that promote homologous recombination at adesired site within the genome, thus providing for intra-chromosomalexpression of these nucleic acids (see e.g. Koller and Smithies, 1989.Proc Natl Acad Sci USA 86: 8932-8935).

Delivery of the nucleic acid as defined above according to the inventioninto a patient for the purpose of gene therapy, particular in thecontext of the above mentioned non-chronic or chronic inflammatory eyediseases as defined above may be either direct (i.e. the patient isdirectly exposed to the nucleic acid or nucleic acid-containing vector)or indirect (i.e. cells are first transformed with the nucleic acid invitro, then transplanted into the patient). These two approaches areknown, respectively, as in vivo or ex vivo gene therapy. In a specificembodiment of the present invention, a nucleic acid is directlyadministered in vivo, where it is expressed to produce the encodedproduct. This may be accomplished by any of numerous methods known inthe art including, e.g. constructing the nucleic acid as part of anappropriate nucleic acid expression vector and administering the same ina manner such that it becomes intracellular (e.g. by infection using adefective or attenuated retroviral or other viral vector; see U.S. Pat.No. 4,980,286); directly injecting naked DNA; using microparticlebombardment (e.g. a “GeneGun”; Biolistic, DuPont); coating the nucleicacids with lipids; using associated cell-surface receptors/transfectingagents; encapsulating in liposomes, microparticles, or microcapsules;administering it in linkage to a peptide that is known to enter thenucleus; or by administering it in linkage to a ligand predisposed toreceptor-mediated endocytosis (see e.g. Wu and Wu, 1987.J Biol Chem 262:4429-4432), which can be used to “target” cell types that specificallyexpress the receptors of interest, etc.

An additional approach to gene therapy in the practice of the presentinvention involves transferring a nucleic acid as defined above intocells in in vitro tissue culture by such methods as electroporation,lipofection, calcium phosphate-mediated transfection, viral infection,or the like. Generally, the method of transfer includes the concomitanttransfer of a selectable marker to the cells. The cells are then placedunder selection pressure (e.g. antibiotic resistance) so as tofacilitate the isolation of those cells that have taken up, and areexpressing, the transferred gene. Those cells are then delivered to apatient. In a specific embodiment, prior to the in vivo administrationof the resulting recombinant cell, the nucleic acid is introduced into acell by any method known within the art including e.g. transfection,electroporation, microinjection, infection with a viral or bacteriophagevector containing the nucleic acid sequences of interest, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer,spheroplast fusion, and similar methods that ensure that the necessarydevelopmental and physiological functions of the recipient cells are notdisrupted by the transfer. See e.g. Loeffler and Behr, 1993. MethEnzymol 217: 599-618. The chosen technique should provide for the stabletransfer of the nucleic acid to the cell, such that the nucleic acid isexpressible by the cell. Preferably, the transferred nucleic acid isheritable and expressible by the cell progeny.

In preferred embodiments of the present invention, the resultingrecombinant cells may be delivered to a patient by various methods knownwithin the art including, e.g. injection of epithelial cells (e.g.subcutaneously), application of recombinant skin cells as a skin graftonto the patient, and intravenous injection of recombinant blood cells(e.g. hematopoietic stem or progenitor cells). The total amount of cellsthat are envisioned for use depend upon the desired effect, patientstate, and the like, and may be determined by one skilled within theart. Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and may bexenogeneic, heterogeneic, syngeneic, or autogeneic. Cell types include,but are not limited to, differentiated cells such as epithelial cells,endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytesand blood cells, or various stem or progenitor cells, in particularembryonic heart muscle cells, liver stem cells (International PatentPublication WO 94/08598), neural stem cells (Stemple and Anderson, 1992,Cell 71: 973-985), hematopoietic stem or progenitor cells, e.g. asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, and the like. In a preferred embodiment, the cells utilized forgene therapy are autologous to the patient.

Alternatively and/or additionally, for treating diseases as mentionedherein targeting therapies may be used to deliver the JNK inhibitorsequences, chimeric peptides, and/or nucleic acids as defined above morespecifically to certain types of cell, by the use of targeting systemssuch as (a targeting) antibody or cell specific ligands. Antibodies usedfor targeting are typically specific for cell surface proteins of cellsassociated with any of the diseases as defined below. By way of example,these antibodies may be directed to cell surface antibodies such as e.g.B cell-associated surface proteins such as MHC class II DR protein, CD18(LFA-1 beta chain), CD45RO, CD40 or Bgp95, or cell surface proteinsselected from e.g. CD2, CD2, CD4, CD5, CD7, CD8, CD9, CD10, CD13, CD16,CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD38, CD39,CD4, CD43, CD45, CD52, CD56, CD68, CD71, CD138, etc. Targetingconstructs may be typically prepared by covalently binding the JNKinhibitor sequences, chimeric peptides, and nucleic acids as definedherein according to the invention to an antibody specific for a cellsurface protein or by binding to a cell specific ligand. Proteins maye.g. be bound to such an antibody or may be attached thereto by apeptide bond or by chemical coupling, crosslinking, etc. The targetingtherapy may then be carried out by administering the targeting constructin a pharmaceutically efficient amount to a patient by any of theadministration routes as defined below, e.g. intraperitoneal, nasal,intravenous, oral and patch delivery routes. Preferably, the JNKinhibitor sequences, chimeric peptides, or nucleic acids as definedherein according to the invention, being attached to the targetingantibodies or cell specific ligands as defined above, may be released invitro or in vivo, e.g. by hydrolysis of the covalent bond, by peptidasesor by any other suitable method. Alternatively, if the JNK inhibitorsequences, chimeric peptides, or nucleic acids as defined hereinaccording to the invention are attached to a small cell specific ligand,release of the ligand may not be carried out. If present at the cellsurface, the chimeric peptides may enter the cell upon the activity ofits trafficking sequence. Targeting may be desirable for a variety ofreasons; for example if the JNK inhibitor sequences, chimeric peptides,and nucleic acids as defined herein according to the invention areunacceptably toxic or if it would otherwise require a too high dosage.

Instead of administering the JNK inhibitor sequences and/or chimericpeptides as defined herein according to the invention directly, theycould be produced in the target cells by expression from an encodinggene introduced into the cells, e.g. from a viral vector to beadministered. The viral vector typically encodes the JNK inhibitorsequences and/or chimeric peptides as defined herein according to theinvention. The vector could be targeted to the specific cells to betreated. Moreover, the vector could contain regulatory elements, whichare switched on more or less selectively by the target cells upondefined regulation. This technique represents a variant of the VDEPTtechnique (virus-directed enzyme prodrug therapy), which utilizes matureproteins instead of their precursor forms.

Alternatively, the JNK inhibitor sequences and/or chimeric peptides asdefined herein could be administered in a precursor form by use of anantibody or a virus. These JNK inhibitor sequences and/or chimericpeptides may then be converted into the active form by an activatingagent produced in, or targeted to, the cells to be treated. This type ofapproach is sometimes known as ADEPT (antibody-directed enzyme prodrugtherapy) or VDEPT (virus-directed enzyme prodrug therapy); the formerinvolving targeting the activating agent to the cells by conjugation toa cell-specific antibody, while the latter involves producing theactivating agent, e.g. a JNK inhibitor sequence or the chimeric peptide,in a vector by expression from encoding DNA in a viral vector (see forexample, EP-A-415731 and WO 90/07936).

According to a further embodiment, the JNK inhibitor sequences, chimericpeptides, nucleic acid sequences or antibodies to JNK inhibitorsequences or to chimeric peptides as defined herein, e.g. an JNKinhibitor sequence according to any of sequences of SEQ ID NOs: 1 to 4and 13 to 20 and 33-100 and/or a chimeric peptide according to any ofsequences of SEQ ID NOs: 9 to 12 and 23 to 32, and/or an JNK inhibitorsequence according to any of sequences of SEQ ID NOs: 1 to 4 and 13 to20 and 33-100 comprising a trafficking sequence according to any of SEQID NOs: 5 to 8 and 21 to 22, or variants or fragments thereof within theabove definitions, may be utilized in (in vitro) assays (e gimmunoassays) to detect, prognose, diagnose, or monitor variousconditions and disease states selected from non-chronic or chronicinflammatory eye diseases as defined above, or monitor the treatmentthereof. The immunoassay may be performed by a method comprisingcontacting a sample derived from a patient with an antibody to an JNKinhibitor sequence, a chimeric peptide, or a nucleic acid sequence, asdefined above, under conditions such that immunospecific-binding mayoccur, and subsequently detecting or measuring the amount of anyimmunospecific-binding by the antibody. In a specific embodiment, anantibody specific for an JNK inhibitor sequence, a chimeric peptide or anucleic acid sequence may be used to analyze a tissue or serum samplefrom a patient for the presence of JNK or a JNK inhibitor sequence;wherein an aberrant level of JNK is indicative of a diseased condition.The immunoassays that may be utilized include, but are not limited to,competitive and non-competitive assay systems using techniques such asWestern Blots, radioimmunoassays (RIA), enzyme linked immunosorbentassay (ELISA), “sandwich” immunoassays, immunoprecipitation assays,precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, fluorescent immunoassays,complement-fixation assays, immunoradiometric assays, and protein-Aimmunoassays, etc. Alternatively, (in vitro) assays may be performed bydelivering the JNK inhibitor sequences, chimeric peptides, nucleic acidsequences or antibodies to JNK inhibitor sequences or to chimericpeptides, as defined above, to target cells typically selected from e.g.cultured animal cells, human cells or micro-organisms, and to monitorthe cell response by biophysical methods typically known to a skilledperson. The target cells typically used therein may be cultured cells(in vitro) or in vivo cells, i.e. cells composing the organs or tissuesof living animals or humans, or microorganisms found in living animalsor humans.

The present invention additionally provides the use of kits fordiagnostic or therapeutic purposes, particular for the treatment,prevention or monitoring of non-chronic or chronic inflammatory eyediseases as defined above, wherein the kit includes one or morecontainers containing JNK inhibitor sequences, chimeric peptides,nucleic acid sequences and/or antibodies to these JNK inhibitorsequences or to chimeric peptides as defined above, e.g. an anti-JNKinhibitor sequence antibody to an JNK inhibitor sequence according toany of sequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100, to achimeric peptide according to any of sequences of SEQ ID NOs: 9 to 12and 23 to 32, to an JNK inhibitor sequence according to any of sequencesof SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 comprising a traffickingsequence according to any of SEQ ID NOs: 5 to 8 and 21 to 22, or to orvariants or fragments thereof within the above definitions, or such ananti-JNK inhibitor sequence antibody and, optionally, a labeled bindingpartner to the antibody. The label incorporated thereby into theantibody may include, but is not limited to, a chemiluminescent,enzymatic, fluorescent, colorimetric or radioactive moiety. In anotherspecific embodiment, kits for diagnostic use in the treatment,prevention or monitoring of non-chronic or chronic inflammatory eyediseases as defined above are provided which comprise one or morecontainers containing nucleic acids that encode, or alternatively, thatare the complement to, an JNK inhibitor sequence and/or a chimericpeptide as defined above, optionally, a labeled binding partner to thesenucleic acids, are also provided. In an alternative specific embodiment,the kit may be used for the above purposes as a kit, comprising one ormore containers, a pair of oligonucleotide primers (e.g. each 6-30nucleotides in length) that are capable of acting as amplificationprimers for polymerase chain reaction (PCR; see e.g. Innis, et al.,1990. PCR PROTOCOLS, Academic Press, Inc., San Diego, Calif.), ligasechain reaction, cyclic probe reaction, and the like, or other methodsknown withinthe art used in context with the nucleic acids as definedabove. The kit may, optionally, further comprise a predetermined amountof a purified JNK inhibitor sequence as defined above, a chimericpeptide as defined above, or nucleic acids encoding these, for use as adiagnostic, standard, or control in the assays for the above purposes.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entirety.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features and advantages of the invention will beapparent from the following detailed description and claims.

DESCRIPTION OF FIGURES

FIG. 1 Clinical efficacy of SEQ ID NO: 11 in LPS-induced uveitis:Clinical scores (expressed in arbitrary units, A.U.) were evaluated atthe peak of the disease, 24 hours after (A) intravenous (IV) or (B)intravitreous (IVT) injection of JNK-inhibitor (poly-)peptide of SEQ IDNO: 11. Comparison was made with untreated uveitic eyes (LPS) and IV/IVTtreatment with vehicle (n=10 eyes per group). Clinical manifestations ofuveitis were reduced after (A) IV injection of JNK-inhibitor(poly-)peptide of SEQ ID NO: 11 (***p<0.001 vs LPS; ###p<0.001 vsVehicle) and (B) IVT injection of (poly-)peptide of SEQ ID NO: 11(***p<0.001 vs LPS; ##p<0.01 vs Vehicle) and dexamethasone (*** p<0.05vs LPS). No statistical difference (ns) was observed between IVTinjection of JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 anddexamethasone that was used as positive control.

FIG. 2 Inhibition of the JNK pathway by SEQ ID NO: 11 in LPS-induceduveitis: Western-Blot analysis of c-Jun phosphorylation inRPE/choroid/sclera complexes 24 hours after IV or IVT injections ofJNK-inhibitor (poly-)peptide of SEQ ID NO: if and vehicle (n=2 eyes pergroup) in Endotoxin-Induced Uveitis (EIU) conditions. Inhibition ofc-Jun phosphorylation by JNK-inhibitor (poly-) peptide of SEQ ID NO: 11was visualized on (A) immunoblot (upper lane: Phospho c-Jun (Ser63);bottom lane: I3-tubulin reporter protein; and confirmed by (B)densitometric quantitation.

FIG. 3 Effects of SEQ ID NO: 11 on the LPS-ERK pathway activation:Immunohistochemistry against phospho-p44/42 MAPK (Erk1/2) (green) wascarried out on ocular histological sections from untreated uveiticcontrol eyes and IV or IVT injected animals (vehicle or SEQ ID NO: 11)(n=3 eyes analyzed per condition; time point: 24 h). Nuclei (in blue)were stained with DAPI. p-Erk1/2 was strongly expressed in the irisepithelium after IVT administration of vehicle (A, a) and SEQ ID NO: 11(B, b), with no detectable difference between the two. Only a faintpositive signal could be detected in retinal mutter glial cells in EIUeyes treated by IV injection of either the vehicle (E, e) or SEQ IDNO:11 (F, f) (see arrows). Scale bar: 100 μm. c: cornea; ir: iris; st:stroma; ep: epithelium; ONH: optic nerve head; INL: inner nuclear layer;ONL: outer nuclear layer.

FIG. 4 Ocular biodistribution of JNK-inhibitor (poly-)peptide of SEQ IDNO: 11 in healthy and uveitic eyes:

Immunohistochemistry against SEQ ID NO: 11 was carried out on ocularhistological sections from untreated and IV or IVT injected animals(vehicle or SEQ ID NO: 11), both in (A-L) healthy and (M-W) LPS-inducedinflammatory conditions (n=3 eyes analyzed per condition; time point: 24h). (A-C) SEQ ID NO: 11 was undetectable in ocular tissues 24 hoursafter IV injection in healthy eyes. (D-L) After IVT injection in healthyeyes, SEQ ID NO: 11 was found in iris epithelium (D, G), ciliary bodyepithelium (E, J), GCL (H), INL (K), IS (I) and RPE (L). In eyes withuveitis, SEQ ID NO: 11 was detected in infiltrating inflammatory cellsafter IV injection of SEQ ID NO: 11 (M-P), but not after IV or IVTinjections of vehicle (not shown) or in untreated EIU eyes (V,W). (Q-U)In uveitic eyes treated by IVT of SEQ ID NO: 11, distribution of SEQ IDNO: 11 was similar to that of healthy treated eyes but was also detectedin migrating resident inflammatory cells (S-T). Scale bar: 50 μm. c:cornea; ir: iris; l: lens; cb: ciliary body; st: stroma; ep: epithelium;GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclearlayer; RPE: retinal pigment epithelium; IS: photoreceptor inner segment;aq. h: aqueous humor.

FIG. 5 Reduction of LPS-induced inflammatory cell infiltration by theJNK-inhibitor (poly-)peptide of SEQ ID NO: 11:

Infiltration of (A) macrophages (ED1 immunopositive cells) and (B)polymorphonuclear leukocytes (PMNs) was quantified on histologicalsections (n=6 sections per group) stained by immunohistochemistry(illustrated on FIG. 6). (A) Intravenous (IV) (**p<0.005 vs LPS) andintravitreous (IVT) (** p<0.005 vs LPS; ##p<0.009 vs Vehicle) injectionsof the (poly-)peptide of SEQ ID NO: 11 reduced the number of ED1+. (B)the (poly-)peptide of SEQ ID NO: 11 decreased the number of PMNs afterintravenous (IV) (**p<0.005 vs LPS) and intravitreous (IVT) (**p<0.005vs LPS; ##p<0.009 vs Vehicle) administrations. No statistical difference(ns) was observed between IVT of the (poly-)peptide of SEQ ID NO: 11 anddexamethasone that was used as a positive control.

FIG. 6 Effect of the (poly-)peptide of SEQ ID NO: 11 on ED1+ cells,polymorphonuclear leukocytes and inducible nitric oxide synthase (iNOS)expression in LPS-induced uveitis:

ED1 and iNOS antigens expression was analyzed by immunohistochemistry oneye cryosections of untreated or treated (IV or IVT, vehicle or SEQ IDNO: 11) uveitic rats (n=3 eyes per condition; time point: 24 h). Nucleiwere stained with DAPI. Numerous inflammatory cells expressing ED1 (A,D, G, J) and/or iNOS (B, E, H, K) infiltrated the anterior (A-I) and theposterior segment (J-L) of untreated EIU eyes. A few number ofED1+/iNOS+ cells were found in the iris/ciliary body (yellow cells,panels c1-2 and fl-2) but most iNOS+ cells were ED-cells suggesting thatmostly PMNs produced iNOS. IV (M-U) and IVT (not shown) injections ofSEQ ID NO:11 reduced the inflammatory infiltrate expressing ED1 (M, P,S) and iNOS (N, Q, T). In eyes treated by IV (M-U) and IVT (not shown)of SEQ ID NO:11, a reduced number of ED1+ cells (M, P, S) and iNOS+cells (N, Q, T) was observed. Scale bar: 50 μm. c: cornea; ir: iris; cb:ciliary body; ret: retina; ON: optic nerve.

FIG. 7 Down-regulation of LPS-induced iNOS expression by the(poly-)peptide of SEQ ID NO: 11:

RT-PCR analysis of inducible nitric oxide synthase (iNOS) mRNA levels inneuroretinas 24 hours after IV or IVT injections of SEQ ID NO:11 orvehicle (n=2 eyes per group) in EIU conditions. Down-regulation of iNOSmRNA was visualized on (A) agarose gel under ultraviolettransilluminator (upper lane: 657 bp iNOS cDNA amplification product;bottom lane: 162 bp GAPDH cDNA amplification product) and confirmed by(B) densitometric quantitation

FIGS. 8A and 8B Modulation of intraocular LPS-induced Chemokine/Cytokineprofiles following intravenous (IV) administration of (poly-)peptide ofSEQ ID NO: 11: Multiplex analysis was performed on ocular fluidscollected 6 h, 24 h and 48 h after EIU induction. Comparison was madebetween uninjected control uveitic rats or with (poly-)peptide of SEQ IDNO: 11 IV treated rats (n=10 eyes analyzed per time point and percondition). Results from rats treated by IV injection of vehicle werenot represented for more clarity. P values of statistical analysis areindicated on each graph (p). IV injection of (poly-)peptide of SEQ IDNO: 11 decreased chemokines production (FIG. 8A), and decreasedpro-inflammatory and Th1 cytokines production at specific time points(FIG. 8B). Levels of IFN-γ and IL-10 were not represented at 48 hbecause of being below detectable levels.

FIGS. 9A and 9B Modulation of intraocular LPS-induced Chemokine/Cytokineprofiles following intravitreous (IVT) administration of (poly-)peptideof SEQ ID NO: 11: Multiplex analysis was performed on ocular fluidscollected 6 h, 24 h and 48 h after EIU induction. Comparison was madebetween rats treated by IVT injection of vehicle and (poly-)peptide ofSEQ ID NO: 11 (n=10 eyes analyzed per time point and per condition). Pvalues of statistical analysis are indicated on each graph (p). Nosignificant changes were observed on chemokines expression betweenvehicle IVT or (poly-)peptide of SEQ ID NO: 11 IVT injections except adecrease of MCP-1 (FIG. 9A). Little changes in cytokines expression wereinduced by IVT injection of the (poly-)peptide of SEQ ID NO: 11: lowerlevels of TNF-α, IL-6 and IL-2 at 6 hours, a lower level of IL-2 and agreater level of IL-13 at 24 hours (FIG. 9A).

FIG. 10 shows the the IB1 cDNA sequence from rat and its predicted aminoacid sequence (SEQ ID NO:102)

FIG. 11 shows the IB1 protein sequence from rat encoded by theexon-intron boundary of the rIB1 gene-splice donor (SEQ ID NO:103)

FIG. 12 shows the IB1 protein sequence from Homo sapiens (SEQ ID NO:104)

FIG. 13 shows the IB1 cDNA sequence from Homo sapiens (SEQ ID NO:105)

EXAMPLES Example 1

Solutions and Products

An all-D-retro-inverso JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 wasproduced by Polypeptide Laboratories (France) and purified by HighPerformance Liquid Chromatography (HPLC). It was analyzed by massspectrometry for identity and RP-HPLC for purity (PolypeptideLaboratories, France). Once lyophilized, the powder was stored at 2-8°C. One day prior to the experiment, the JNK-inhibitor (poly-)peptide ofSEQ ID NO: 11 powder was dissolved under sterile conditions at theconcentration of 10 μM in saline (NaCl 0.9%, Versol®, Aguettant) in aNational Scientific (NSC) deactivated glass vial (NSC-C4015-S1) andstored at 4° C. until use.

For each experiment, a fraction of freshly dissolved the JNK-inhibitor(poly-)peptide of SEQ ID NO: 11 was stored at −20° C. and itsconcentration was confirmed by High Performance Liquid Chromatography(HPLC) analysis.

Dexamethasone sodium phosphate 4 mg/mL (Soludecadron; LaboratoireRoussel, Paris, France) was used as positive control for antiinflammatory activity on EIU.10

Animals

7 weeks old female Lewis rats weighing 175 g (Elevage Janvier, Le GenestSaint Isle, France) were used and handled in accordance with the ARVOStatement for the Use of Animals in Ophthalmic and Vision Research. Ratswere anesthetized with intramuscular injection of Ketamine (88 mg/kg)(Virbac, France) and Largactil (0.6 mg/kg) (Sanofi-Aventis, France)before intravenous or ocular injection.

Injections

For intravenous (IV) injection, 100 μL of saline (NaCl 0.9%) or theJNK-inhibitor (poly-)peptide of SEQ ID NO: 11 (20 μg/kg in saline) wereinjected in a tail vein using a 25G-needle connected on a 1 mL syringe(Becton Dickinson, France). For intravitreous (IVT) injection, 5 μL ofsaline or the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 (0.2μg/injection in saline) were injected in both eyes using a 30Gdisposable needle (BD-microfine syringe, nm Médical, Asnière, France).The IV dose of 20 μg/kg (i.e. 3.5 μg/rat in rats weighing 175 g) waschosen according to studies showing that the JNK-inhibitor(poly-)peptide of SEQ ID NO: 11 is active at very low doses in othermodels. For intravitreous injections, the inventors used the minimaldose used in direct ear application after acute noise trauma inpatients. This corresponds to 5% of the dose injected intravenously.Immediately after intravenous or intravitreous treatment,Endotoxin-Induced Uveitis (EIU) was induced by a single footpadinjection of 100 μL sterile pyrogen-free saline containing 200 μg of LPS(Lipopolysaccharides from Salmonella typhimurium, Sigma-Aldrich,Saint-Quentin Fallavier, France). At the end the experiments, i.e. 6, 24or 48 h after LPS challenge, rats were anesthetized by intraperitonealinjection of pentobarbital (30 mg/kg) (Sanofi-Aventis, France) beforeblood was collected by intracardiac puncture. Rats were then killed witha lethal dose of pentobarbital and both eyes were enucleated.

Samples Collection

Aqueous humor and vitreous were collected and pooled from eachenucleated eye. Ocular fluids were immediately centrifuged and thecell-free fractions were collected and frozen at −20° C. before analysisby Multiplex assay. Blood samples were first clotted at room temperaturefor 2 hours and then at 4° C. overnight. Serum was collected,centrifuged and the clear supernatant was collected and frozen at −20°C. before Multiplex analysis.

Retinas and RPE/choroid/sclera complexes were carefully dissected out onenucleated eyes, snap frozen and stored at −80° C. until being used forRT-PCR and Western-Blot analyses.

For immunohistochemistry, eyeballs were collected and fixed for 1 h atroom temperature in phosphate buffered saline (PBS) containing 4%paraformaldehyde before being rinsed overnight in PBS. The next day,samples were embedded and frozen in optimal cutting-temperature (OCT)compound (Tissue-Tek®, Sakura Finetek, Zoeterwoude, Netherland) andstored at −80° C. Frozen antero-posterior sections of eyes (10 μm thick)were performed at the optic nerve level using a cryostat (Leica CM3050S, Rueil-Malmaison, France) and mounted on super-frost slides forimmunohistochemical analysis.

Experimental Design

In a first set of experiment, 70 rats were randomized into 14experimental groups with 5 rats per group. Uveitis was induced in eachgroup and rats were killed 6 hours (4 groups), 24 hours (6 groups) and48 hours (4 groups) after LPS challenge. For each time point tested(i.e. 6 h, 24 h and 48 h), rats treated by intravenous or intravitreousinjections of vehicle (NaCl 0.9%) or the JNK-inhibitor (poly-)peptide ofSEQ ID NO: 11 were compared to untreated control uveitic rats. Twoadditional groups, treated by intravenous injection of vehicle andintravitreal injection of dexamethasone were used at 24 hours. Clinicalocular inflammation was recorded only at 24 hours (see Scoring ofEndotoxin-Induced Uveitis (EIU) section). At each time point,intraocular fluids from each eye (n=10 per group) and serum from eachanimal (n=5 per group) were used for Chemokine/Cytokine Multiplex Assay.

Retinas and RPE/choroid/sclera complexes were also collected at 24 hoursto analyze iNOS mRNA levels by RT-PCR and c-Jun phosphorylation state byWestern-Blot. Tissues were collected only from eyes treated by IV(intravenous) injection of vehicle, IV injection of the JNK-inhibitor(poly-)peptide of SEQ ID NO: 11, IVT (intravitreal) injection of vehicleand IVT injection of the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11(n=2 eyes per condition collected from separate rats). Eyes wereselected so that their EIU clinical score was representative of the meanof the experimental group they belong to, i.e 3 for eyes treated by IVand IVT injection of vehicle and 2 for eyes treated by IV or IVTinjection of the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11.

A second set of experiment was designed to evaluate theanti-inflammatory effect of the JNK-inhibitor (poly-)peptide of SEQ IDNO: 11 at the cellular and tissue level as well as the biodistributionof this molecule 24 hours after administration. Rats were randomizedinto 11 experimental groups. 6 groups of rats with uveitis: untreateduveitic rats, rats injected intravenously with NaCl or the JNK-inhibitor(poly-)peptide of SEQ ID NO: 11 and rats injected intravitreously withthe vehicle, the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 ordexamethasone. The 5 additional groups without uveitis were: untreatedhealthy rats, rats treated by NaCl IV or the JNK-inhibitor(poly-)peptide of SEQ ID NO: 11 IV and rats injected IVT with NaCl orthe JNK-inhibitor (poly-)peptide of SEQ ID NO: 11. Three eyes fromseparate rats were collected per group and used forimmunohistochemistry.

Note that, for clinical and histological analyses, dexamethasone wasused as a reference treatment.

Scoring of Endotoxin-Induced Uveitis (EIU)

Animals were examined by slit lamp at 24 hours, the clinical peak of thedisease in our experiments. The intensity of clinical ocularinflammation was scored on a scale from 0 to 5 for each eye as describedpreviously10: grade (0) indicates no inflammation; grade (1) indicatesthe presence of a minimal iris and conjunctival vasodilation but withoutthe observation of flare or cells in the anterior chamber (AC); grade(2) indicates the presence of moderate iris and conjunctival vesseldilation but without evident flare or cells in the AC; grade (3)indicates the presence of intense iris vessels dilation, flare and lessthan 10 cells per slit lamp field in the AC; grade (4) indicates thepresence of more severe clinical signs than grade 3, with more than 10cells in the AC with or without the formation of a hypopion; grade (5)indicates the presence of intense inflammatory reaction, fibrinformation in the AC and total seclusion of the pupil. Clinicalevaluation was performed in a masked manner.

Western-Blot Analysis

RPE/choroid/sclera complexes and neuroretinas (2 per experimental group)were snap frozen immediately after dissection and stored at −80° C.until use. Tissues were homogenized in 500 μL, of lysis buffer (MOPS SDSRunning Buffer, Invitrogen, Cergy-Pontoise, France) supplemented withprotease inhibitor cocktail (Roche Diagnostics, Meylan, France) (onetablet for 50 mL). After addition of LDS Sample Buffer (Invitrogen) andheating for 5 min at 100° C., equal amounts of proteins were subjectedto electrophoresis in a NuPAGE 4-12% Bis-Tris gel (Invitrogen) usingMOPS SDS Running Buffer. The bands obtained were then electrotransferredonto nitrocellulose membranes (Schleicher & Schnell BioScience, Dassel,Germany).

Western-blot analyses were carried out to analyze the effect of theJNK-inhibitor (poly-)peptide of SEQ ID NO: 11 on the threemitogen-activated protein kinase (MAPK) pathways. To analyze the JNKpathway, blots were sequentially incubated with a rabbit Phospho-c-Jun(Ser63) primary antibody (or Phospho-c-Jun (Ser73) antibody) and ananti-rabbit IgG HRP-linked secondary antibody according to themanufacturer's instruction (PhosphoPlus c-Jun (Ser63) II and c-Jun(Ser73) antibody kit (9260) purchased from Cell Signaling Technology(Ozyme, St Quentin Yvelines, France)). Bands were visualized using theECL Western Blotting Detection Reagents Kit (Amersham Biosciences,Orsay, France). Blots were then dehybridized and rehybridizedsuccessively with a mouse anti □-tubulin (D-10) (sc-5274) primaryantibody (dilution 1:400) and a HRP conjugated goat anti-mouse IgGsecondary antibody (sc-3697) (dilution 1:5000) (both purchased fromSanta Cruz Biotechnology (Tebu-bio, Le Perray en Yvelines Cedex,France)). The relative band intensity for phospho c-Jun (Ser 63 orSer73) was calculated in comparison to that for □-tubulin afterdensitometry analysis (ImageJ software).

To analyze the ERK and p38 MAPK pathways, blots were sequentiallyincubated with a rabbit phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)(4370) primary antibody (or rabbit phospho-p38 MAPK (Thr180/Tyr182)(9215) antibody) and a horseradish peroxidase-conjugated goatanti-rabbit IgG (H+L) (PI-1000—Vector Laboratories, Clinisciences,Montrouge, France) secondary antibody at dilution 1:5000. Blots werethen dehybridized and rehybridized successively with a rabbit p44/42MAPK (Erk1/2) (4695) (or rabbit p38 MAP Kinase (9212) antibody) and thesame secondary antibody as above. Primary antibodies were purchased fromCell Signaling Technology (Ozyme, St Quentin Yvelines, France) and allsteps performed following the manufacturer's instruction.

Immunohistochemistry

To characterize the cellular infiltrate, sections were double-stainedwith ED1 and iNOS. Briefly, after permeabilization with 0.1% TritonX-100in phosphate buffered saline (PBS) for 30 min, specimens were rinsed andsaturated for 30 min with 5% skimmed milk in PBS. They were incubatedovernight at 4° C. with the two following primary antibodies: a 1:50mouse monoclonal anti-macrosialin CD68 (clone ED1), directed against acytoplasmic antigen in rat monocytes, macrophages and dendritic cells(purchased from Serotec Ltd. (Oxford, UK)) and a polyclonal rabbitanti-iNOS (1/75e; Transduction Laboratories, Lexinton, FY). Afterwashing, sections were incubated for 1 hour at room temperature with asecondary Alexa Fluor 594 (red)-conjugated donkey anti-mouse monoclonalantibody (mAb) and a secondary Alexa Fluor 488 (green)-conjugated goatanti-rabbit mAb each at dilution 1:250 (Invitrogen, Cergy Pontoise,France). For each step, antibodies were diluted in PBS-1% skimmedmilk—0.1% TritonX100. Different controls were included in every stainingrun: negative controls without primary antibodies and isotype controlsby incubation with normal mouse or rabbit serum immunoglobulin (Ig) inplace of primary antibodies. After staining nuclei with DAPI(Sigma-Aldrich, Saint-Quentin Fallavier, France), sections were mountedin PBS/Glycerol (1/1) and observed by fluorescence photomicroscopy (FXA,Microphot, Nikon, Melville, USA). Digitized micrographs were obtainedusing a digital camera (Spot, BFI Optilas, Evry, France). ED1 positivecells and polymorphonuclear cells, identified by the shape of theirnuclei stained with DAPI, were quantified on histological sections. Theanalysis was performed on 3 eyes per experimental group, with 2different sections per eye at the optic nerve head level. Results wereexpressed as mean±standard error of the mean (SEM).

Immunostaining of the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 wasperformed on healthy and uveitic eyes to study its ocularbiodistribution after systemic or local administration. Briefly,sections were permeabilized as described above before being sequentiallyincubated with an anti-SEQ ID NO: 11 purified rabbit IgG and a secondaryAlexa 594 (red)-conjugated goat anti-rabbit IgG (Invitrogen, CergyPontoise, France) diluted 1:100 and 1:250 in PBS respectively.Immunostaining of untreated healthy and uveitic eyes were used asnegative controls. Nuclei were stained with DAPI before mounting andobservation.

Immunostaining of p-Erk1/2 was performed to evaluate the effect of theJNK-inhibitor (poly-) peptide of SEQ ID NO: 11 on the ERK pathway afterIV or IVT administration. Sections were permeabilized as described aboveand incubated in blocking solution containing 0.1% Triton X-100 and 10%FCS (fetal calf serum) in PBS for 1 hour at room temperature. Sectionswere incubated overnight at 4° C. with a rabbit anti-phospho-p44/42 MAPK(Erk1/2) primary antibody (4370) purchased from Cell SignalingTechnology (Ozyme, St Quentin Yvelines, France) diluted 1:400 inblocking solution. After having been rinsed three times in PBS, sectionswere incubated with a secondary Alexa Fluor 488 (green)-conjugated goatanti-rabbit mAb (diluted 1:300 in blocking solution) for 2 hours at roomtemperature. Nuclei were stained with DAPI before mounting andobservation.

Evaluation of iNOS Expression in Ocular Tissues Using Semi-QuantitativePCR

Two eyes per group were used for this analysis. Immediately afterdissection, retinas extracted from each eye were separately snap frozenand stored at −80° C. until use. Total RNA was extracted from tissues(RNeasy minikit, Qiagen, Courtaboeuf, France) according to themanufacturer's instructions. Reverse transcription was performed on 1 μgof total RNA in a total volume of 20 μL using Superscript II ReverseTranscriptase (Invitrogen, Cergy-Pontoise, France) following themanufacturer's instructions. To amplify GAPDH and iNOS cDNA,Polymerase-Chain Reaction (PCR) was conducted in a total volume of 25 μLcontaining 2 μL of first-strand reaction product, 0.4 μM forward and 0.4μM reverse primers, 0.4 μM dNTP Mix, 1.5 mM MgCl2, 1×PCR buffer and 2.5U Taq DNA polymerase (Invitrogen, Cergy Pontoise, France). Primersspecific for GAPDH (Forward: 5′-ATGCCCCCATGTTTGTGATG-3′ (SEQ ID NO:101);Reverse: 5′-ATGGCATGGACTGTGGTCAT-3′ (SEQ ID NO:106)) and iNOS (Forward:5′-TTTCTCTTCAAAGTCAAATCCTACCA-3′ (SEQ ID NO:107); Reverse:5′-TGTGTCTGCAGATGTGCTGAAAC-3′ (SEQ ID NO:108)) were obtained fromInvitrogen. After an initial denaturation (3 min at 94° C.), 30 to 32PCR cycles of denaturation (30 s, 94° C.), annealing (1 min, 58° C.(GAPDH) and 52° C. (iNOS)) and elongation (1 to 2 min, 72° C.) wereperformed on a Crocodile III (Appligene Oncor). The final cycle wascompleted by 5 min of elongation at 72° C. PCR fragments (162 bp forGAPDH and 657 bp for iNOS) were analyzed by 2.5% agarose gelelectrophoresis and visualized by ethidium bromide staining under UVlight. The relative band intensity for iNOS was calculated in comparisonto that for GAPDH after densitometry analysis (ImageJ software).

Chemokine/Cytokine Multiplex Assay

Intraocular fluids (diluted to obtain a final volume of 25 μL) and sera(25 μL of 1:5 dilution) were subjected to multiplex bead analysis. Thismethod uses microspheres as the solid support for immunoassays12 andallows the titration of a greater number of cytokines with increasedsensitivity than occurs with ELISA.13 For each sample, seventeenanalytes were quantified simultaneously using the ratCytokine/Chemokine-17plex kit (Milliplex Map Kit, Millipore,Saint-Quentin-en-Yvelines, France) according to the manufacturer'sinstructions: Chemokines MCP-1/CCL2, MIP-1α/CCL3, RANTES/CCL5,IP-10/CXCL10 (IFN-inducible protein-10) and GRO/KC; proinflammatorymediators IL-1□, IL-18 and TNF-□; Th1/Th2/Th17 cytokines IL-2 andIFN-□/IL-4, IL-5, IL-6, IL-10 and IL-13/IL-17. The assay was performedin a 96-well filter plate and standard curves for each cytokine weregenerated with a Rat Cytokine Standard provided in the kit. Allincubation steps were performed under medium orbital agitation and inthe dark to protect the beads from light. Data acquisition and analysiswere performed with the manager software version 4.1 (Bioplex; Bio-Rad)with four or five logistic parameters for standard curves. Detectionthresholds for all the analytes were estimated around 1 to 10 pg/mL.

Statistical Analysis

Numerical results were expressed as mean±standard error of the mean(SEM). Data were compared using the nonparametric Mann-Whitney U-test.P<0.05 was considered statistically significant.

Results:

The all-D-retro-inverso JNK-inhibitor (poly-)peptide of SEQ ID NO: 11significantly reduced endotoxins induced uveitis (EIU). TheJNK-inhibitor (poly-)peptide of SEQ ID NO: 11 significantly reduced EIUclinical scores after 20 μg/kg intravenous (IV) injection (2.0±0.1)compared to untreated uveitic eyes (3.2±0.1, p<0.001) and vehicle IV(3.2±0.1, p<0.001) (FIG. 1A). In a similar manner, clinical scores weresignificantly decreased after 0.2 μg/injection intravitreous (IVT)administration of the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11(2.2±0.2) in comparison to untreated uveitic eyes (3.2±0.1, p<0.001) andvehicle IVT (3.0±0.1, p<0.01) (FIG. 1B). The effect of IVT injection ofthe JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 on clinical signs ofEIU was not statistically different from that observed after IVT ofdexamethasone (1.8±0.4) suggesting that the JNK-inhibitor (poly-)peptideof SEQ ID NO: 11 was as efficient as dexamethasone in reducing EIU whenadministered at this time point (FIG. 1B).

Efficacy of the (Poly-)Peptide of SEQ ID NO: 11 Resulted from JNKPathway Inhibition

To determine whether the clinical effect of the JNK-inhibitor(poly-)peptide of SEQ ID NO: 11 was related to its mode of action, i.e.its ability to interfere with JNK signaling, 6 c-Jun phosphorylationstate was analyzed in ocular tissues by Western-Blot. Phosphorylation ofc-Jun on Ser63 (FIG. 2A) and Ser73 residues was reduced in RPE/choroidextracts 24 hours after the JNK-inhibitor (poly-)peptide of SEQ ID NO:11 was injected intravenously or intravitreously. In the neuroretina,phospho c-Jun could only be faintly detected. An approximately 3-folddecrease in c-Jun phosphorylation was observed in RPE/choroid eitherafter IV (0.28±0.01 vs 0.77±0.26 in IV of vehicle) or IVT (0.35±0.08 vs0.79±0.25 in IVT of vehicle) administration of the JNK-inhibitor(poly-)peptide of SEQ ID NO: 11 (FIG. 2B). The ability of theJNK-inhibitor (poly-)peptide of SEQ ID NO: 11 to block c-JunNH2-terminal kinases (JNK) activity in the eye tissues demonstrated thespecific intraocular activity of the JNK-inhibitor (poly-)peptide of SEQID NO: 11.

To determine whether the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11could have any effect on the other MAPK pathways, the phosphorylationstate of Erk1/2 and p38 was evaluated. Whereas Erk1/2 and p38 weredetected in RPE/choroid complexes at similar levels among all groups,the phosphorylation form of these two MAPK could not be detected bywestern-blot analysis (data not shown). These results demonstrate thatJNK is the predominantly activated MAPK pathway in RPE/choroid duringEIU. Using histochemical analysis, performed without any signalamplification, we found an intense p-Erk1/2 signal in inflammatory cellsinfiltrating in the anterior and the posterior segments of the eye inthe control LPS and saline treated eyes (FIGS. 3C, 3D). The effect ofthe JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 either administeredintravenously or intravitreously could not be evaluated on those cells,since the infiltration was almost absent in the treated eyes. However,in the neuroretina, where p-Erk1/2 could be detected and located inretinal Müller glial (RMG) cells in the control and saline treated eyes(FIG. 3E), no effect of the JNK-inhibitor (poly-)peptide of SEQ ID NO:11 administered by either route was observed (FIG. 3F). Interestingly,in the iris, an intense p-Erk1/2 signal was observed in the epitheliumof the control and saline injected eyes (FIG. 3A) with no effect of theJNK-inhibitor (poly-)peptide of SEQ ID NO: 11 treated on the p-Erk1/2signal in these cells (FIG. 3B), strongly suggesting that theJNK-inhibitor (poly-)peptide of SEQ ID NO: 11 does not seem to directlyact on p-Erk1/2 phosphorylation during EIU in our model, at least inresident cells.

Differential Distribution of the JNK-Inhibitor (Poly-)Peptide of SEQ IDNO: 11 in Ocular Tissues after IV and IVT Administrations

Immunohistochemistry was carried out on histological sections toevaluate the biodistribution of the JNK-inhibitor (poly-)peptide of SEQID NO: 11 in ocular tissues 24 hours after systemic (IV) or local (IVT)administration, both in healthy eyes and in uveitic conditions (FIG. 4).No inflammatory cell infiltration was observed in healthy eyes eitherafter IV or IVT of the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 orvehicle. No immunoreactivity against the JNK-inhibitor (poly-)peptide ofSEQ ID NO: 11 was detected in untreated control eyes or in eyes treatedby vehicle, demonstrating the specificity of the signal observed in theJNK-inhibitor (poly-)peptide of SEQ ID NO:11-treated eyes. Whereas nosignal was observed in normal eyes after systemic (IV) injection (FIG. 4A-C) at the dose used, the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11was distributed in almost all ocular tissues of normal rats after IVTadministration (FIG. 4 D-L). Interestingly, an accumulation of theJNK-inhibitor (poly-) peptide of SEQ ID NO: 11 was detected mainly inthe iris/ciliary body epithelium (panels G and J) and in the retinalpigment epithelium (panel L). Penetration of the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 was also detected in the iris stroma (panel G)as well as in the neural retina in the ganglion cell layer (GCL, panelH), the inner nuclear layer (INL, panel K) and the inner segment (IS,panel I) of photoreceptor cells (PR). In all cell types, theJNK-inhibitor (poly-) peptide of SEQ ID NO: 11 accumulated within thecytoplasm. Occasional staining was found in the corneal endothelium andin the lens capsule.

In uveitic conditions, no the JNK-inhibitor (poly-)peptide of SEQ ID NO:11 staining was detected in ocular tissues and in infiltratinginflammatory cells of untreated eyes (FIG. 4, panels V-W). IV or IVT ofvehicle gave similar results to those from untreated eyes. In EIU eyestreated by IV injection of the JNK-inhibitor (poly-)peptide of SEQ IDNO: 11, it was not detected in ocular tissues, but occasionalinfiltrating inflammatory cells were immunopositive in the iris (panelO) and in the aqueous humor (panel P). In uveitic eyes treated by IVTinjection, the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 was mostlyfound in ocular tissues like in healthy eyes and in resident cells thatare mobilized and participate actively to the inflammatory processes inpathological conditions such as microglial cells (panels S-T).

A Significant Reduction in Cells Infiltrating the Ocular TissuesResulted from the JNK-Inhibitor (Poly-)Peptide of SEQ ID NO: 11Administration

To further characterize the effect of the JNK-inhibitor (poly-)peptideof SEQ ID NO: 11 in uveitis, the infiltrated inflammatory cells werequantified in ocular tissues (FIG. 5) by numeration on histologicalsections immunostained with ED1 and iNOS antibodies (FIG. 6). 24 hoursafter LPS challenge, the number of ED1 positive cells was significantlyreduced in eyes treated with IV injection of the JNK-inhibitor(poly-)peptide of SEQ ID NO: 11 (137±7) (FIG. 5A, FIG. 6M, P, S) ascompared to untreated uveitic eyes (LPS) (187±13, p<0.005) (FIG. 5A,FIG. 6A, D, G, J) or vehicle injected eyes. Similarly, IVT of theJNK-inhibitor (poly-) peptide of SEQ ID NO: 11 significantly reduced ED1positive infiltrating cells (93±8) as compared to vehicle IVT injectedeyes (175±15, p<0.009) and untreated uveitic eyes (p<0.005) (FIG. 5A).The reducing effect of the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11on ED positive cell infiltration (93±8) did not differ from that inducedby dexamethasone (79±15), suggesting that both treatments have a similarefficacy on this parameter.

The number of polymorphonuclear cells (PMN) (FIG. 5B) was alsosignificantly reduced at 24 hours after IV administration of theJNK-inhibitor (poly-)peptide of SEQ ID NO: 11 (60±6) as compared tocontrol eyes (237±15, p<0.005), and after IVT injection of theJNK-inhibitor (poly-)peptide of SEQ ID NO: 11 (40±5) as compared to IVTinjection of the vehicle (152±31, p<0.009) and control uveitic eyes(p<0.005). Again, the effect of the JNK-inhibitor (poly-)peptide of SEQID NO: 11 on PMN ocular tissue infiltration did not significantly differfrom that of dexamethasone (42±11).

The JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 down regulates iNOSexpression Since iNOS (inducible nitric oxide synthase) has beendescribed as a key mediator in the pathogenesis of uveitis,14,15 theeffect of the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 on itsexpression was investigated at both the protein and mRNA levels.

As shown on FIG. 6, the number of iNOS positive cells was reduced ineyes treated with injection of the JNK-inhibitor (poly-)peptide of SEQID NO: 11 IV (FIG. 6N, Q, T) or IVT compared to control eyes (panels BE, H, K). Among iNOS positive cells observed in control eyes, a fewnumber were ED1+ cells while most of them were ED1-suggesting thatmostly PMNs produced iNOS (insets c, f, i). In eyes from theJNK-inhibitor-treated rats, the only cells still expressing iNOS wereintra tissular ED1 positive cells located at the ciliary body root(inset r2).

The effect of the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 on iNOSexpression was confirmed by RT-PCR on ocular tissues (FIG. 7). Levels ofiNOS mRNA were down-regulated from 1.02±0.21 to 0.40±0.11 after IV ofthe JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 and from 1.18±0.05 to0.27±0.09 in eyes treated by IVT injection. Comparisons were made withIV or IVT of vehicle respectively.

Chemokine/Cytokine Profiles in Ocular Media of Eyes Treated with theJNK-Inhibitor (Poly-)Peptide of SEQ ID NO: 11

To evaluate the effect of the treatment on the production of pro- andanti-inflammatory mediators, chemokines and cytokines were dosed bymultiplex analysis on ocular media (FIGS. 8 and 9) and sera.

Among the 17 chemokines/cytokines tested, some were below detectablelevels both in control or treated eyes: IP-10, IL-5, IL-17. Other didnot differ in treated versus untreated eyes at any of the tested timepoints: IL-18, IL-4, IL-1β. In the serum, while some cytokines tended tochange after IV administration of the JNK-inhibitor (poly-)peptide ofSEQ ID NO: 11 (reduction of MIP-1α and IL-2) or after IVT (reduction ofIL-2), this was not statistically significant. For the otherchemokines/cytokines, their profile was different in ocular fluids fromeyes treated with IV administration of the JNK-inhibitor (poly-)peptideof SEQ ID NO: 11 as compared to the IVT administration. Indeed, when theJNK-inhibitor (poly-)peptide of SEQ ID NO: 11 was injected systemicallyat the time of LPS challenge, it induced a significant reduction ofMCP-1, MIP-1α and RANTES at 6 and 24 hours (FIG. 8A). GRO/KC was alsosignificantly reduced at 6 hours. Th1 cytokines such as TNF-α, IL-6 andINF-γ were significantly reduced at different time points while IL-10tended to increase at 6 hours in treated eye (but not significantly),suggesting a switch towards a Th2 profile (FIG. 8B). No statisticaldifferences were noticed between eyes from vehicle IV injected rats anduntreated uveitic control eyes.

When the JNK-inhibitor (poly-)peptide of SEQ ID NO: 11 was injected intothe vitreous at the time of LPS challenge, the chemokine/cytokineprofiles was not strikingly different from that of eyes injected withvehicle (FIGS. 9A and 9B). It is interesting to note though that IVT ofvehicle had a marked effect on the cytokine profile as compared tountreated uveitic control eyes. At 6 hours, a trend to a decrease ofMCP-1, TNF-α, IL-6, IL-2 and at 24 hours, a marked decrease of IL-2 andan increase of IL-13 were detected suggesting again a switch towards aTh2 profile.

The invention claimed is:
 1. A method for treating a chronic or non-chronic inflammatory eye disease in a subject in need thereof, the method comprising administering a JNK inhibitor (poly-) peptide comprising less than 150 amino acids in length to the subject, wherein the chronic or non-chronic inflammatory eye disease is selected from the group consisting of posterior uveitis, an inflammatory eye disease of the blephera, an inflammatory eye disease of the conjunctiva, an an inflammatory eye disease of the sclera, an inflammatory eye disease of the vitreous body, and an inflammatory eye disease of the orbital bone.
 2. The method of claim 1, wherein the JNK inhibitor (poly-)peptide comprises SEQ ID NO:
 11. 